Method for preparing flour doughs and products made from such doughs using glycerol oxidase

ABSTRACT

Method of improving the rheological properties of a flour dough and the quality of bread, alimentary paste products, noodles and cakes wherein glycerol oxidase or a combination of glycerol oxidase and a lipase is added to the dough and dough improving compositions comprising these enzymes. The strength of (B/C ratio) and the gluten index of the dough was improved and in the resulting products the improvements were higher specific volume, increased crumb pore homogeneity and reduced average crumb pore diameter.

FIELD OF THE INVENTION

[0001] The present invention relates to the field of food manufacturing,in particular to the preparation of improved bakery products and otherfarinaceous food products. Specifically, the invention concerns the useof glycerol oxidase as a dough strengthening agent and improvement ofthe quality of baked and dried products made from such improved doughs.There is also provided a method of improving the properties of doughsand baked product by combined use of glycerol oxidase and a lipase.

TECHNICAL BACKGROUND AND PRIOR ART

[0002] The “strength” or “weakness” of doughs is an important aspect ofmaking farinaceous finished products from doughs, including baking. The“strength” or “weakness” of a dough is primarily determined by itscontent of protein and in particular the content and the quality of thegluten protein is an important factor in that respect. Flours with a lowprotein content is generally characterized as “weak”. Thus, thecohesive, extensible, rubbery mass which is formed by mixing water andweak flour will usually be highly extensible when subjected to stress,but it will not return to its original dimensions when the stress isremoved.

[0003] Flours with a high protein content are generally characterized as“strong” flours and the mass formed by mixing such a flour and waterwill be less extensible than the mass formed from a weak flour, andstress which is applied during mixing will be restored without breakdownto a greater extent than is the case with a dough mass formed from aweak flour. Strong flour is generally preferred in most baking contextsbecause of the superior rheological and handling properties of the doughand the superior form and texture qualities of the finished baked ordried products made from the strong flour dough.

[0004] Doughs made from strong flours are generally more stable.Stability of a dough is one of the most important characteristics offlour doughs. Within the bakery and milling industries it is known touse dough “conditioners” to strengthen the dough to increase itsstability and strength. Such dough conditioners are normallynon-specific oxidizing agents such as e.g. iodates, peroxides, ascorbicacid, K-bromate or azodicarbonamide and they are added to dough with theaims of improving the baking performance of flour to achieve a doughwith improved stretchability and thus having a desirable strength andstability. The mechanism behind this effect of oxidizing agents is thatthe flour proteins, in particular gluten contains thiol groups which,when they become oxidized, form disulphide bonds whereby the proteinforms a more stable matrix resulting in a better dough quality andimprovements of the volume and crumb structure of the baked products.

[0005] However, the use of several of the currently availablenon-specific oxidizing agents is either objected to by consumers or isnot permitted by regulatory bodies. Hence it has been attempted to findalternatives to these conventional flour and dough additives, and theprior art has i.a. suggested the use of glucose oxidase and hexoseoxidase for this purpose.

[0006] Glycerol oxidase is an oxidoreductase which is capable ofoxidizing glycerol. Different types of glycerol oxidase have beendescribed in the literature. Some of these glycerol oxidases needco-factors in order to oxidize glycerol (Shuen-Fu et al., 1996. EnzymeMicro. Technol., 18:383-387).

[0007] However, glycerol oxidase from Aspergillus japonicus does notrequire any co-factors in the oxidation of glycerol to glyceraldehyde(T. Uwajima and O. Terada, 1980. Agri. Biol. Chem. 44:2039-2045).

[0008] This glycerol oxidase has been characterized by T. Uwajima and O.Terada (Methods in Enzymology, 1982, 89:243-248) and T. Uwajima et al.(Agric. Biol. Chem., 1979, 43:2633-2634), and has a pH optimum at 7.0and K_(m) and V_(max) are 10.4 mM and 935.6 μmol H₂O₂ min⁻¹ respectivelyusing glycerol as substrate. The enzyme is most active on glycerol butalso other substrates like dihydroxyacetone, 1,3-propanediol,D-galactose ad D-fructose are oxidized by glycerol oxidase.

[0009] Glycerol oxidase not requiring co-factors has also been isolatedfrom Penicillium and characterized by Shuen-Fuh Lin et al. (EnzymeMicro. Technol., 1996, 18:383-387). This enzyme has optimum activity inthe pH range from 5.5 to 6.5 at 30° C. The enzyme is stable between 20and 40° C. but loses its activity at temperatures above 50° C.

[0010] Other potential sources for glycerol oxidase according to theinvention include different fungal species as disclosed in DE-2817087-A,such as Aspergillus oryzae, Aspergillus parasiticus, Aspergillus flavus,Neurospora crassa, Neurospora sitophila, Neurospora tetrasperma,Penicillium nigricans, Penicillium funiculosum and Penicilliumjanthinellum.

[0011] Glycerol oxidase isolated from the above natural sources has beenused for different applications. Thus, glycerol oxidase from Aspergillusjaponicus has been used for glycoaldehyde production from ethyleneglycol (Kimiyasu Isobe and Hiroshi Nishise, 1995, Journal of MolecularCatalysis B: Enzymatic, 1:37-43). Glycerol oxidase has also been used inthe combination with lipoprotein lipase for the determination ofcontaminated yolk in egg white (Yioshinori Mie, 1996. Food ResearchInternational, 29:81-84). DE-2817087-A and U.S. Pat. No. 4,399,218disclose the use of glycerol oxidase for the determination of glycerol.

[0012] It has now been found that the addition of a glycerol oxidase toa flour dough results in an increased resistance hereof to deformationwhen the dough is stretched, i.e. this enzyme confers to the dough anincreased strength whereby the dough becomes less prone to mechanicaldeformation. Accordingly, glycerol oxidase is highly useful as a doughconditioning agent in the manufacturing of flour dough based productsincluding not only bread products but also other products made fromflour doughs such as noodles and alimentary paste products.

[0013] It has also been found that the dough strengthening effect ofglycerol oxidase is potentiated significantly when it is combined with alipase, which in itself does not affect the dough strength. Furthermore,the combined use of glycerol oxidase and lipase results in animprovement of bread quality, in particular in respect of specificvolume and crumb homogeneity, which is not a simple additive effect, butreflects a synergistic effect of these two types of enzymes.

SUMMARY OF THE INVENTION

[0014] Accordingly, the invention relates in a first aspect to a methodof improving the rheological properties of a flour dough and the qualityof the finished product made from the dough, comprising adding to thedough 10 to 10,000 units of a glycerol oxidase per kg of flour.

[0015] In a further aspect there is provided a method of improving therheological properties of a flour dough and the quality of the finishedproduct made from the dough, comprising adding to the dough a glyceroloxidase and a lipase.

[0016] The invention pertains in a still further aspect to doughimproving composition comprising a glycerol oxidase and at least onefurther dough ingredient or dough additive.

[0017] In still further aspects, the invention relates to the use of aglycerol oxidase for improving the rheological properties of a flourdough and the quality of the finished product made from the dough and tothe use of a glycerol oxidase and a lipase in combination for improvingthe rheological properties of a flour dough and the quality of thefinished product made from the dough.

DETAILED DISCLOSURE OF THE INVENTION

[0018] In one aspect, the present method provides a method of improvingthe rheological properties of flour doughs.

[0019] The expression “rheological properties” as used herein refersparticularly to the effects of dough conditioners on dough strength andstability as the most important characteristics of flour doughs.According to American Association of Cereal Chemists (AACC) Method36-01A the term “stability” can be defined as “the range of dough timeover which a positive response is obtained and that property of arounded dough by which it resists flattening under its own weight over acourse of time”. According to the same method, the term “response” isdefined as “the reaction of dough to a known and specific stimulus,substance or set of conditions, usually determined by baking it incomparison with a control”

[0020] As it is mentioned above, it is generally desirable to improvethe baking performance of flour to achieve a dough with improvedstretchability and thus having a desirable strength and stability byadding oxidizing agents which cause the formation of protein disulphidebonds whereby the protein forms a more stable matrix resulting in abetter dough quality and improvements of the volume and crumb structureof baked products.

[0021] Thus, the term “rheological properties” relates to the abovephysical and chemical phenomena which in combination will determine theperformance of flour doughs and thereby also the quality of theresulting products.

[0022] The method comprises, as it is mentioned above, the addition ofan effective amount of a glycerol oxidase to the dough. It will beunderstood that the addition can be either to a component of the doughrecipe or to the dough resulting from mixing all of the components forthe dough. In the present context, “an effective amount” is used toindicate that the amount is sufficient to confer to the dough and/or thefinished product improved characteristics as defined herein.Specifically, such an amount is in the range of 10 to 10,000 units ofglycerol oxidase per kg flour.

[0023] In one useful embodiment of the method according to theinvention, the glycerol oxidase can, as it is described in detailsherein, be isolated from a bacterial species, a fungal species, a yeastspecies, an animal cell including a human cell or a plant cell. Examplesof glycerol oxidase producing fungal species are species belonging tothe genera Aspergillus, Neurospora and Penicillium, such as A.japonicus, A. oryzae, A. parasiticus, A. flavus, Neurospora crassa, N.sitophila, N. tetrasperma, Penicillium nigricans, P. funiculosum and P.janthinellum.

[0024] Glycerol oxidase can be derived as a native enzyme from naturalsources such as the above.

[0025] It is one objective of the invention to provide improved bakeryproducts. In accordance with the invention, a bakery product doughincluding a bread dough is prepared by mixing flour with water, aleavening agent such as yeast or a conventional chemical leaveningagent, and an effective amount of glycerol oxidase under dough formingconditions. It is, however, within the scope of the invention thatfurther components can be added to the dough mixture.

[0026] Typically, such further dough components include conventionallyused dough components such as salt, sweetening agents such as sugars,syrups or artificial sweetening agents, lipid substances includingshortening, margarine, butter or an animal or vegetable oil, glyceroland one or more dough additives such as emulsifying agents, starchdegrading enzymes, cellulose or hemicellulose degrading enzymes,proteases, lipases, non-specific oxidizing agents such as thosementioned above, flavouring agents, lactic acid bacterial cultures,vitamins, minerals, hydrocolloids such as alginates, carrageenans,pectins, vegetable gums including e.g. guar gum and locust bean gum, anddietary fiber substances.

[0027] Conventional emulsifying agents used in making flour doughproducts include as examples monoglycerides, diacetyl tartaric acidesters of mono- and diglycerides of fatty acids, and lecithins e.g.obtained from soya. Among starch degrading enzymes, amylases areparticularly useful as dough improving additives. Other useful starchdegrading enzymes which may be added to a dough composition includeglucoamylases and pullulanases. In the present context, furtherinteresting enzymes are xylanases and oxidoreductases such as glucoseoxidase, pyranose oxidase, hexose oxidase, sulfhydryl oxidase, andlipases.

[0028] A preferred flour is wheat flour, but doughs comprising flourderived from other cereal species such as from rice, maize, barley, ryeand durra are also contemplated.

[0029] In accordance with the invention, the dough is prepared byadmixing flour, water, the glycerol oxidase and optionally otheringredients and additives. The glycerol oxidase can be added togetherwith any dough ingredient including the water or dough ingredientmixture or with any additive or additive mixture. The dough can beprepared by any conventional dough preparation method common in thebaking industry or in any other industry making flour dough basedproducts.

[0030] The glycerol oxidase can be added as a liquid preparation or inthe form of a dry powder composition either comprising the enzyme as thesole active component or in admixture with one or more other doughingredients or additive.

[0031] The amount of the glycerol oxidase added is an amount whichresults in the presence in the dough of 10 to 5,000 units (as defined inthe following) such as 10 to 2,500 units per kg of flour. In usefulembodiments, the amount is in the range of 20 to 1,500 units per kg offlour.

[0032] The effect-of the glycerol oxidase on the rheological propertiesof the dough can be measured by standard methods according to theInternational Association of Cereal Chemistry (ICC) and the AmericanAssociation of Cereal Chemistry (AACC) including the amylograph method(ICC 126), the farinograph method (AACC 54-21) and the extensigraphmethod (AACC 54-10). The AACC method 54-10 defines the extensigraph inthe following manner: “the extensigraph records a load-extension curvefor a test piece of dough until it breaks. Characteristics ofload-extension curves or extensigrams are used to assess general qualityof flour and its responses to improving agents”. In effect, theextensigraph method measures the relative strength of a dough. A strongdough exhibits a higher and, in some cases, a longer extensigraph curvethan does a weak dough.

[0033] In a preferred embodiment of the method according to theinvention, the resistance to extension of the dough in terms of theratio between the resistance to extension (height of curve, B) and theextensibility (length of curve, C), i.e. the B/C ratio as measured bythe AACC method 54-10 is increased by at least 10% relative to that ofan otherwise similar dough not containing glycerol oxidase. In morepreferred embodiments, the resistance to extension is increased by atleast 20%, such as at least 50% and in particular by at least 100%.

[0034] It has been found that the addition of glycerol oxidase to bakeryproduct doughs results in bakery products such as yeast leavened andchemically leavened products in which the specific volume is increasedrelative to an otherwise similar bakery product, prepared from a doughnot containing glycerol oxidase. In this context, the expression“specific volume” is used to indicate the ratio between volume andweight of the product. It has been found that, in accordance with theabove method, the specific volume can be increased significantly such asby at least 10%, preferably by at least 20%, including by at least 30%,preferably by at least 40% and more preferably by at least 50%.

[0035] The method according to the invention is highly suitable forimproving the rheological properties and quality of the finishedproducts of conventional types of yeast leavened bread products based onwheat flour, such as loaves and rolls. The method is also suitable forimproving the rheological properties of doughs containing chemicalleavening agents (baking powder) and the quality of products made fromsuch doughs. Such product include as examples sponge cakes and muffins.

[0036] In one interesting aspect, the invention is used to improve therheological properties of doughs intended for noodle products including“white noodles” and “chinese noodles” and to improve the texturalqualities of the finished noodle products. A typical basic recipe forthe manufacturing of noodles comprises the following ingredients: wheatflour 100 parts, salt 0.5 parts and water 33 parts. Furthermore,glycerol is often added to the noodle dough. The noodles are typicallyprepared by mixing the ingredients in an appropriate mixing apparatusfollowed by rolling out the noodle dough using an appropriate noodlemachine to form the noodle strings which are subsequently air dried.

[0037] The quality of the finished noodles is assessed i.a. by theircolour, cooking quality and texture. The noodles should cook as quicklyas possible, remain firm after cooking and should preferably not looseany solids to the cooking water. On serving the noodles shouldpreferably have a smooth and firm surface not showing stickiness andprovide a firm “bite” and a good mouthfeel. Furthermore, it is importantthat the white noodles have a light colour.

[0038] Since the appropriateness of wheat flour for providing noodleshaving the desired textural and eating qualities may vary according tothe year and the growth area, it is usual to add noodle improvers to thedough in order to compensate for sub-optimal quality of the flour.Typically, such improvers will comprise dietary fiber substances,vegetable proteins, emulsifiers and hydrocolloids such as e.g.alginates, carrageenans, pectins, vegetable gums including guar gum andlocust bean gum, and amylases, and as mentioned above, glycerol.

[0039] It is therefore an important aspect of the invention that theglycerol oxidase according to the invention is useful as a noodleimproving agent optionally in combination with glycerol and othercomponents currently used to improve the quality of noodles. Thus, it iscontemplated that noodles prepared in accordance with the above methodwill have improved properties with respect to colour, cooking and eatingqualities including a firm, elastic and non-sticky texture andconsistency.

[0040] In a further useful embodiment, the dough which is prepared bythe method according to the invention is a dough for preparing analimentary paste product. Such products which include as examplesspaghetti and maccaroni are typically prepared from a dough comprisingmain ingredients such as flour, eggs or egg powder and/or water. Aftermixing of the ingredient, the dough is formed to the desired type ofpaste product and air dried. It is contemplated that the addition ofglycerol oxidase to a paste dough, optionally in combination withglycerol, will have a significant improving effect on the extensibilityand stability hereof resulting in finished paste product having improvedtextural and eating qualities.

[0041] In a useful embodiment, there is provided a dough improvingmethod wherein at least one further enzyme is added to the doughingredient, dough additive or the dough. In the present context,suitable enzymes include cellulases, hemicellulases, xylanases, starchdegrading enzymes, oxidoreductases and proteases.

[0042] In a further aspect, the invention relates to a method ofimproving the rheological properties of a flour dough and the quality ofthe finished products made from the dough which comprises that both aglycerol oxidase and a lipase is added to the dough.

[0043] It was surprisingly found that the two types of enzymes werecapable of interacting with each other under the dough conditions to anextent where the effect on improvement of the dough strength and breadquality by the enzymes was not only additive, but the effect wassynergistic.

[0044] Thus, with respect to improvement of dough strength it was foundthat with glycerol oxidase alone, the B/C ratio as measured after 45minutes of resting was increased by 34%, with lipase alone no effect wasobserved. However, when combining the two enzymes, the B/C ratio wasincreased by 54%, i.e. combining the glycerol oxidase with the lipaseenhanced the dough strengthening effect of glycerol oxidase by more than50%. Thus, one objective of combining glycerol oxidase and a lipase isto provide an enhancement of the dough strengthening effect of glyceroloxidase by at least 25% such as at least 50% including at least 75%,determined as described herein.

[0045] In relation to improvement of finished product, it was found thatthe combined addition of glycerol oxidase and a lipase resulted in asubstantial synergistic effect in respect to crumb homogeneity asdefined herein. Also, with respect to the specific volume of bakedproduct a synergistic effect was found. Thus, for a bread product, theaddition of lipase alone typically results in a negligible increase ofthe specific volume, addition of glycerol oxidase alone in an increaseof about 25%, whereas a combined addition of the two enzymes results inan increase of more than 30%.

[0046] Further in relation to improvement of the finished product, itwas found that the addition of lipase resulted in modification of theglycolipids, monogalactosyl diglyceride and digalactosyl diglyceridepresent in dough. These components were converted to the more polarcomponents monogalactosyl monoglyceride and digalactosyl monoglyceride.As galactosyl monoglycerides are more surface active components thangalactosyl diglycerides it is assumed that galactosyl monoglyceridescontributed to the observed improved crumb cell structure andhomogeneity. Thus, one objective of using lipase is to hydolyse at least10% of the galactosyl diglycerides normally present in a flour dough tothe corresponding galactosyl monoglycerides, such as at least 50%including at least 100%.

[0047] The details of such a method using combined addition of glyceroloxidase and lipase are, apart from the use of a lipase in combinationwith glycerol oxidase, substantially similar to those described abovefor a method according to the invention which does not require theaddition of a lipase.

[0048] When using, in accordance with the invention, a lipase incombination with a glycerol oxidase, the amount of lipase is typicallyin the range of 10 to 100,000 lipase units (LUS) (as defined in thefollowing) per kg flour including the range of 10 to 20,000 LUS, e.g.100 to 15,000 LUS such as 500 to 10,000 LUS.

[0049] Lipases that are useful in the present invention can be derivedfrom a bacterial species, a fungal species, a yeast species, an animalcell and a plant cell. Whereas the enzyme may be provided by cultivatingcultures of such source organisms naturally producing lipase, it may bemore convenient and cost-effective to produce it by means of geneticallymodified cells such as it is described in details in the followingexamples. In the latter case, the term “derived” may imply that a genecoding for the lipase is isolated from a source organism and insertedinto a host cell capable of expressing the gene.

[0050] Thus, the enzyme may in a useful embodiment be derived from anAspergillus species including as examples A. tubigensis, A. oryzae andA. niger.

[0051] Presently preferred lipases include the lipase designated Lipase3, the production and characteristics of which is described in detailsin the following examples, or a mutant of this enzyme. In the presentcontext, the term “mutant” refers to a lipase having, relative to thewild-type enzyme, an altered amino acid sequence. A further preferredlipase is the lipase found in the commercial product, GRINDAMYL™ EXEL16.

[0052] In a further aspect of the invention there is provided a doughimproving composition comprising a glycerol oxidase and at least onefurther dough ingredient or dough additive.

[0053] The further ingredient or additive can be any of the ingredientsor additives which are described above. The composition may convenientlybe a liquid preparation comprising the glycerol oxidase. However, thecomposition is conveniently in the form of a dry composition.

[0054] The amount of the glycerol oxidase in the composition is in therange of 10 to 10,000 units per kg flour. It will be appreciated thatthis indication of the amount of enzyme implies that a recommendedappropriate amount of the composition will result in the above statedamount in the dough to which it is added. In specific embodiments, theamount of glycerol oxidase is in the range of 10 to 5,000 units such as10 to 2,500 units per kg of flour. In other useful embodiments, theamount is in the range of 20 to 1,500 units per kg of flour.

[0055] In another embodiment, the dough improving composition mayfurther comprises a lipase as defined above and in the amounts as alsodescribed above in relation to the method according to the invention.

[0056] Optionally, the composition is in the form of a complete doughadditive mixture or pre-mixture for making a particular finished productand containing all of the dry ingredients and additives for such adough. In specific embodiments, the composition is one particularlyuseful for preparing a baking product or in the making of a noodleproduct or an alimentary paste product.

[0057] In one advantageous embodiment of the above method at least onefurther enzyme is added to the dough. Suitable examples hereof include acellulase, a hemicellulase, a xylanase, a starch degrading enzyme,hexose oxidase and a protease.

[0058] In a preferred advantageous embodiment, the further added enzymeis a lipase. It has been found that in accordance with the above method,the crumb homogeneity and specific volume of the bakery product can beincreased significantly as compared to that of an otherwise similarbakery product prepared from a dough not containing glycerol oxidase,and from a similar bakery product prepared from a dough containingglycerol oxidase.

[0059] In a still further aspect, the present invention pertains to theuse of a glycerol oxidase and a lipase in combination for improving therheological properties of a flour dough and the quality of the finishedproduct made from the dough.

[0060] In this connection, specific embodiments include use wherein theimprovement of the rheological properties of the dough include that theresistance to extension of the dough in terms of the ratio betweenresistance to extension (height of curve, B) and the extensibility(length of curve, C), i.e. the B/C ratio, as measured by the AACC method54-10 is increased by at least 10% relative to that of an otherwisesimilar dough that does not contain glycerol oxidase and use wherein theimprovement of the quality of the finished product made from the doughis that the average pore diameter of the crumb of the bread made fromthe dough is reduced by at least 10%, relative to a bread which is madefrom a bread dough without addition of the lipase.

[0061] In a further embodiment, the use according to the invention,implies that the improvement of the quality of the finished product madefrom the dough consists in that the pore homogeneity of the crumb of thebread made from the dough is increased by at least 5%, relative to abread which is made from a bread dough without addition of the lipase.The pore homogeneity of bread is conveniently measured by means of animage analyzer composed of a standard CCD-video camera, a videodigitiser and a personal computer with WinGrain software. Using such ananalyzer, the results of pore diameter in mm and pore homogeneity can becalculated as an average of measurements from 10 slices of bread. Thepore homogeneity is expressed in % of pores that are larger than 0.5times the average of pore diameter and smaller than 2 times the averagediameter.

[0062] In a further embodiment, the use relates to improvement of therheological characteristics of the dough including that the gluten index(as defined hereinbelow) in the dough is increased by at least 5%,relative to a dough without addition of a lipase, the gluten index isdetermined by means of a Glutomatic 2200 apparatus.

BRIEF DESCRIPTION OF THE FIGURES

[0063] The present invention is further illustrated by reference to theaccompanying figures in which

[0064]FIG. 1 shows the restriction map of the genomic clone of the lipAgene,

[0065]FIG. 2 shows the structure of the lipA gene encoding lipase 3,

[0066]FIG. 3 shows a chromatogram of HIC fractionated culturesupernatant of an Aspergillus tubigensis transformant with 62-foldincrease of lipase 3, and

[0067]FIG. 4 shows a chromatogram of HIC fractionated culturesupernatant of the untransformed Aspergillus tubigensis strain.

[0068] The invention will now be described by way of illustration in thefollowing non-limiting examples.

[0069] A. Production and Purification of Glycerol Oxidase (GLOX)

EXAMPLE 1 Production, Extraction and Purification of Glycerol OxidaseUsing Different Strains and Cultivation Conditions

[0070] 1. Production, Extraction and Purification of Glycerol OxidaseUsing Aspergillus japonicus ATCC 1042 Cultivated in a Production MediumContaining 3% Glycerol

[0071] The following assay for determination of glycerol oxidaseactivity was used:

[0072] The assay is based on the method described by Sullivan and Ikawa(Biochimica and Biophysica Acta, 1973, 309:11-22), but modified asdescribed in the following. An assay mixture containing 150 μl 2%glycerol (in 100 mM phosphate buffer, pH 7.0), 120 μl 100 mM phosphatebuffer, pH 7.0, 10 μl o-dianisidin dihydrochloride (Sigma D 3252, 3mg/ml in H₂O), 10 μl peroxidase (POD) (Sigma P8125, 0.1 mg/ml in 100 mMphosphate buffer, pH 7.0) and 10 μl glycerol oxidase (GLOX) solution.The controls are made by adding buffer in place of GLOX solution. Theincubation is started by the addition of glycerol. After 15 minutes ofincubation at 25° C. in microtiter plates, the absorbance at 402 nm isread in a Elisa reader. A standard curve is constructed using varyingconcentrations of H₂O₂ in place of the enzyme solution. The reaction canbe described in the following manner:

[0073] Oxidised o-dianisidine has a yellow colour absorbing at 402 nm.

[0074] One glycerol oxidase unit (U) is the amount of enzyme whichcatalyses the production of 1 82 mole H₂O₂ per minute at 25° C., pH 7.0at a substrate concentration of 0.2 M glycerol.

[0075] A spore suspension of Aspergillus japonicus ATCC 1042 wasprepared by incubating A.japonicus on PDA medium (30° C., 7 days) andwashing with 10 ml of 0.2% Tween 80. A preculture was prepared byinoculating 1 ml of the resulting spore suspension in 300 ml productionmedium containing 3.0% of glycerol (87%, Merck), 0.3% of yeast extract(Difco), 0.1% of meat extract (Difco), 0.1% KH₂PO₄ (Merck), 0.1% ofMGSO₄*7H₂O (Merck), 0.1% antifoam (Contra spum) and 70 mg/l ofchloramphenicolum (Mecobenzon) (pH adjusted to 7.2 with NaOH) in a 500ml flask. The preculture was incubated overnight at 30° C. with shaking(200 rpm).

[0076] A 30 litre fermenter with 15 litre production medium wasinoculated with 900 ml (corresponding to 3 flasks) of the resultingovernight preculture, and cultured at 30° C. for 25 hours undercontinuous stirring (350 rpm) and aeration (15 1/min). After culturing,the mycelia was harvested from the resulting culture broth by filtrationon a Whatman GF/B filter by suction, and washed with 3 litres ofdeionized water. The mycelium yield was 186 g (wet weight).

[0077] A part (50 g) of the resulting mycelial mat was suspended in 700ml of 50 mM borate buffer (pH 10.0), and disrupted by ultrasonication(Branson, Sonifer 250) at 5° C. (3×5 minutes). After disruption, themycelia was removed by centrifugation (29,000 g for 15 minutes), thecell-free extract (700 ml) was brought to 40% saturation with ammoniumsulfate and the resulting precipitate was removed by centrifugation(29,000 g for 20 minutes). The ammonium sulfate concentration was thenincreased to 70% saturation to precipitate the enzyme. The resultingprecipitate was collected and solubilized in 100 ml of 50 mM boratebuffer (pH 10.0). The crude extract was then dialysed for 24 hoursagainst 5 1 of 50 mM borate buffer (pH 10.0). After dialysis theinsoluble matters in the crude extract were removed by centrifugation(18,000×g for 10 minutes). The resulting supernatant contained 8.7 unitsof glycerol oxidase activity per ml.

[0078] 2. Production, Extraction and Purification of Glycerol OxidaseUsing Aspergillus japonicus ATCC 1042 Cultivated in a Production MediumContaining 5% Glycerol

[0079] A spore suspension of Aspergillus japonicus ATCC 1042 wasprepared as described above. A preculture was prepared by inoculating 1ml of the resulting spore suspension into a flask (500 ml) containing200 ml production medium (5.0% glycerol, 0.25% yeast extract, 0.1% Maltextract, 0.7% anti-foam (Contra spum), pH adjusted to 6.2 with HCl,sterilization at 121° C. for 90 minutes). The preculture was incubated 3days at 30° C. with continuous shaking (200 rpm). A 6 litre fermenterwith 5 litre production medium as described above was inoculated with 50ml of the resulting preculture and cultured at 30° C. for 3 days undercontinuous stirring (250 rpm) and aeration (5 1/min). After culturingthe mycelia was harvested from the resulting culture broth by filtrationon a Whatman GF/B filter by suction, and washed with 3 litre ionizedwater containing 0.9% NaCl.

[0080] The resulting mycelia mat was frozen in liquid nitrogen,suspended in 200 ml of 50 mM phosphate buffer (pH 7.0) and disrupted byultrasonication (Branson, Sonifer 250) at 5° C. (4 minutes). Afterdisruption, the mycelia was removed by filtration on a Whatman GF/Afilter by suction. The enzyme in the resulting filtrate was concentratedon a AMICON® 8400 ultrafiltration unit and contained 87 units ofglycerol oxidase per ml after ultrafiltration.

[0081] 3. Production, Extraction and Purification of Glycerol OxidaseUsing Aspergillus japonicus ATCC 1042 Cultivated in a Production MediumContaining 10% Glycerol

[0082] A spore suspension of Aspergillus japonicus ATCC 1042 wasprepared as described above. A 1 ml sample of the resulting sporesuspension was inoculated into each of 5 flasks (500 ml) with 200 mlproduction medium containing 10.0% of glycerol, 0.1% of yeast extractand 0.1% of malt extract (pH adjusted to 6.2 with HCl, sterilization at121° C. for 15 minutes). The cultures were incubated for 5 days at 30°C. with shaking (140 rpm).

[0083] The extraction and concentration of the enzyme was carried out asdescribed above. The resulting filtrate contained 66 units of glyceroloxidase per ml after ultrafiltration.

[0084] 4. Production of Glycerol Oxidase from Penicillium funiculosumand Penicillium janthinellum

[0085] Spore suspensions of Penicillium funiculosum NRRL 1132 andPenicillium janthinellum NRRL 2016 were prepared as described above. A 1ml sample of each of the resulting spore suspensions was inoculated intoseparate flasks (1000 ml) containing 100 g wheat bran and 100 ml water(two flasks for each culture)

[0086] Glycerol oxidase was extracted by suspending the wheat brancultures in 900 ml of 30 mM phosphate buffer (pH 6.5) containing 0.1%Triton X100 (Merck). The mycelial mat was removed from the cultivationmedia by filtration using a Whatman GF/B filter. The resulting myceliamat was frozen in liquid nitrogen, suspended in 200 ml of 50 mMphosphate buffer (pH 7.0) and disrupted by ultrasonication (Branson,Sonifer 250) at 5° C. (4 minutes). After disruption, the mycelia wasremoved by filtration on a Whatman GF/A filter by suction. The resultingfiltrate from the Penicillium funiculosum culture contained 7.4 units ofglycerol oxidase per ml, and the resulting filtrate from the Penicilliumjanthinellum culture contained 11.3 units of glycerol oxidase per ml.

[0087] B. Production, Purification and Characterization of Aspergillustubigensis LIPASE 3

[0088] Materials and Methods

[0089] (i) Determination of lipase activity and protein

[0090] 1. Plate Assay on Tributyrin-containing Medium

[0091] The assay is modified from Kouker and Jaeger (Appl. Environ.Microbiol., 1987, 53:211-213).

[0092] A typical protocol for this assay is as follows: 100 ml 2% agarin 50 mM sodium phosphate buffer (pH 6.3) is heated to boiling, andafter cooling to about 70° C. under stirring, 5 ml 0.2% Rhodamine B isadded under stirring plus 40 ml of tributyrin. The stirring is continuedfor 2 minutes. The mixture is then sonicated for 1 minute. After anadditional 2 minutes of stirring, 20 ml of the agar mixture is pouredinto individual petri dishes. In the absence of lipase activity, theagar plates containing tributyrin and Rhodamine B will appear opaque andare pink coloured.

[0093] To quantify lipase activity, holes having a diameter of 3 mm arepunched in the above agar and filled with 10 μl of lipase preparation.The plates are incubated for varying times at 37° C. When lipaseactivity is present in the applied preparation to be tested, a sharppink/reddish zone is formed around the holes. When the plates areirradiated with UV light at 350 nm, the lipase activity is observed ashalos of orange coloured fluorescence.

[0094] 2. Modified Food Chemical Codex Assay for Lipase Activity

[0095] Lipase activity based on hydrolysis of tributyrin is measuredaccording to Food Chemical Codex, Forth Edition, National Academy Press,1996, p. 803. With the modification that the pH is 5.5 instead of 7. OneLUT (lipase unit tributyrin) is defined as the amount of enzyme whichcan release 2 μmol butyric acid per min. under the above assayconditions.

[0096] 3. p-nitrophenyl Acetate Assay

[0097] Lipase activity can also be determined colorimetrically usingp-nitrophenyl acetate as a substrate e.g. using the following protocol:In a microtiter plate 10 μl of sample or blank is added followed by theaddition of 250 μl substrate (0.5 mg p-nitrophenyl acetate per ml 50 mMphosphate buffer, pH 6.0).

[0098] The microtiter plate is incubated for 5 minutes at 30° C. and theabsorbance at 405 nm is read using a microplate reader. 1 unit isdefined as 1 μmol p-nitrophenol released per 5 minutes.

[0099] 4. p-nitrophenyl Hexanoate Assay

[0100] Lipase activity can be determined by using p-nitrophenylhexanoate as a substrate. This assay is carried out by adding 10 μl ofsample preparation or blank to a microtiter plate followed by theaddition of 250 μl substrate (0.5 mg p-nitro-phenyl hexanoate per ml of20 mM phosphate buffer, pH 6.). At this concentration of substrate thereaction mixture appears as a milky solution. The microtiter plate isincubated for 5 minutes at 30° C. and the absorbance at 405 nm is readin a microplate reader.

[0101] 5. Titrimetric Assay of Lipase Activity

[0102] Alternatively, lipase activity is determined according to FoodChemical Codex (3rd Ed., 1981, pp 492-493) modified to sunflower oil andpH 5.5 instead of olive oil and pH 6.5. The lipase activity is measuredas LUS (lipase units sunflower) where 1 LUS is defined as the quantityof enzyme which can release 1 μmol of fatty acids per minute fromsunflower oil under the above assay conditions.

[0103] 6. Protein Measurement

[0104] During the course of purification of lipase as described in thefollowing, the protein eluted from the columns was measured bydetermining absorbance at 280 nm. The protein in the pooled samples wasdetermined in microtiter plates by a sensitive Bradford method accordingto Bio-Rad (Bio-Rad Bulletin 1177 EG, 1984). Bovine serum albumin wasused as a standard.

EXAMPLE 2 Production, Purification and Characterization of Lipase 3

[0105] 2.1. Production

[0106] A mutant strain of Aspergillus tubigensis was selected and usedfor the production of wild type lipase. This lipase is referred toherein as lipase 3. The strain was subjected to a fermentation in a 7501 fermenter containing 410.0 kg of tap water, 10.8 kg soy flour, 11.1 kgammonium monohydrogenphosphate, 4.0-kg phosphoric acid (75%), 2.7 kgmagnesium sulfate, 10.8 kg sunflower oil and 1.7 kg antifoam 1510. Thesubstrate was heat treated at 121° C. for 45 minutes. The culture mediawas inoculated directly with 7.5×10⁹ spores of the mutant strain. Thestrain was cultivated for three days at 38° C., pH controlled at 6.5,aeration at 290 1/min and stirring at 180 rpm the first two days and at360 rpm the last day. The fermentate was separated using a drum filterand the culture filtrate was concentrated 3.8 times by ultra-filtration.The concentrated filtrate was preserved with potassium sorbate (0.1%)and sodium benzoate (0.2%) and used as a starting material forpurification of lipase.

[0107] 2.2. Purification of Lipase

[0108] A 60 ml sample of ferment (cf. 2.1) containing 557 LUS/ml, pH 5.5was first filtered through a GF/B filter and subsequently through a 0.45μm filter. The filtered sample was desalted using a Superdex G25 SPcolumn (430 ml, 22×5 cm) equilibrated in 20 mM triethanolamine, pH 7.3.The flow rate was 5 ml/min. The total volume after desalting was 150 ml.

[0109] The desalted sample was applied to a Source Q30 anion exchangercolumn (100 ml, 5×5 cm) equilibrated in 20 mM triethanolamine, pH 7.3.The column was washed with equilibration buffer until a stable baselinewas obtained.

[0110] Lipase activity was eluted with a 420 ml linear gradient from 0to 0.35 M sodium chloride in equilibration buffer, flow rate 5 ml/min.Fractions of 10 ml were collected. Sodium acetate (100 μl of a 2Msolution) was added to each fraction to adjust pH to 5.5. Fractions26-32 (70 ml) were pooled.

[0111] To the pool from the anion exchange step was added ammoniumsulfate to 1 M and the sample was applied to a Source Phenyl HIC column(20 ml, 10×2 cm) equilibrated in 20 mM sodium acetate (pH 5.5), 1 Mammonium sulfate. The column was washed with the equilibration buffer.Lipase was eluted with a 320 ml linear gradient from 1 M to 0 M ammoniumsulfate in 20 mM sodium acetate (pH 5.5), flow 1.5 ml/min. Fractions of7.5 ml were collected.

[0112] Fractions 33-41 were analyzed by SDS-PAGE using a NOVEX systemwith precast gels. Both electrophoresis and silver staining of the gelswere done according to the manufacturer (Novex, San Diego, USA). (Thesame system was used for native electrophoresis and isoelectricfocusing). It was found that fraction 40 and 41 contained lipase as theonly protein.

[0113] 2.3. Characterization of the Purified Lipase

[0114] (i) Determination of molecular weight

[0115] The apparent molecular weight of the native lipase was 37.7 kDaas measured by the above SDS-PAGE procedure. The purified lipase elutedat a molecular weight of 32.2 kDa from a Superose 12 gel filtrationcolumn (50 mM sodium phosphate, 0.2 M sodium chloride, pH 6.85, flow0.65 ml/min) and is therefore a monomer.

[0116] The molecular weight of the lipase was also determined bymatrix-assisted laser desorption ionisation (MALDI) by means of atime-of-flight (TOF) mass spectrometer (Voyager Bio-SpectrometryWorkstation, Perspective Biosystems). Samples were prepared by mixing0.7 μl of desalted lipase solution and 0.7 μl of a matrix solutioncontaining sinapic acid (3.5-dimethoxy-4-hydroxy cinnamic acid) in 70%acetonitrile (0.1% TFA, 10 mg/ml). 0.7 μl of the sample mixture wasplaced on top of a stainless steel probe tip and allowed to air-dryprior to introduction into the mass spectrometer. Spectra were obtainedfrom at least 100 laser shots and averaged to obtain a good signal tonoise ratio. The molecular mass for the lipase was found to be 30,384 Daand 30,310 Da by two independent analyses.

[0117] Digestion of the lipase with endo-p-N-acetyl-glucosamidase H (10μl) from Streptomyces (Sigma) was carried out by adding 200 μl lipaseand incubating at 37° C. for 2 hours. The digestion mixture was desaltedusing a VSWP filter and analyzed directly by MALDI mass spectrometry. Amajor component of deglycosylated lipase gave a mass of 29,339 Da and29,333 Da by two independent analyses. A minor component with a mass of29,508 Da was also observed. These values corresponds well to the latercalculated theoretical value of 28,939 Da based on the complete aminoacid sequence of the mature lipase.

[0118] (ii) Determination of the isoelectric point

[0119] The isoelectric point (pI) for the lipase was determined byisoelectric focusing and was found to be 4.1.

[0120] A calculation of the pI based on the amino acid sequence asdetermined in the following and shown as SEQ ID NO: 9 gave an estimatedpI of 4.07.

[0121] (iii) Determination of temperature stability

[0122] Eppendorf tubes with 25 μl of purified lipase 3 plus 50 μl 100 mMsodium acetate buffer (pH 5.0) were incubated for 1 hour in a water bathat respectively 30, 40, 50, and 60° C. A control was treated in the sameway, but left at room temperature. After 1 hour the lipase 3 activitywas determined by the p-nitrophenyl acetate assay as described above.

[0123] The purified lipase had a good thermostability. It was found thatthe lipase maintained 60% of its activity after 1 hour at 60° C. 80% and85% activity was maintained after 1 hour at 50° C. and 40° C.respectively.

[0124] (iv) Determination of pH stability

[0125] Purified lipase 3 (200 μl) was added to 5 ml of 50 mM buffersolutions: (sodium phosphate, pH 8.0, 7.0 and 6.0 and sodium acetate pH5.0, 4.0 and 3.5). The control was diluted in 5 ml of 4 mM sodiumacetate pH 5.5. After four days at room temperature the residualactivity was measured by the Modified Food Chemical Codex assay forlipase activity as described above. The lipase was very stable in the pHrange from 4.0 to 7.0 where it maintained about 100% activity relativeto the control (Table 2.1). At pH 3.5 the lipase maintained 92%activity, and at pH 8.0 95% residual activity was maintained as comparedto the control. TABLE 2.1 pH stability of lipase 3 pH Activity (LUT/ml)Activity (%) Control (pH 5.5) 89.2 100 3.5 82.5 92 4.0 91.7 103 5.0 86.597 6.0 92.4 104 7.0 90.6 102 8.0 84.4 95

EXAMPLE 3 Amino Acid Sequencing of Lipase 3

[0126] Purified lipase enzyme was freeze-dried and 100 μg of thefreeze-dried material was dissolved in 50 μl of a mixture of 8 M ureaand 0.4 M ammonium hydrogencarbonate, pH 8.4. The dissolved protein wasdenatured and reduced for 15 minutes at 50° C. following overlay withnitrogen and addition of 5 μl 45 mM dithiothreitol. After cooling toroom temperature, 5 μl of 100 mM iodoacetamide was added for thecysteine residues to be derivatized for 15 minutes at room temperaturein the dark under nitrogen.

[0127] 135 μl of water and 5 μg of endoproteinase Lys-C in 5 μl of waterwas added to the above reaction mixture and the digestion was carriedout at 37° C. under nitrogen for 24 hours. The resulting peptides wereseparated by reverse phase HPLC on a VYDAC C18 column (0.46×15 cm; 10μm; The Separation Group, California, USA) using solvent A: 0.1% TFA inwater and solvent B: 0.1% TFA in acetonitrile. Selected peptides wererechromatographed on a Develosil C18 column (0.46×10 cm, Novo Nordisk,Bagsvmrd, Denmark) using the same solvent system, prior to N-terminalsequencing. Sequencing was done using an Applied Biosystems 476Asequencer using pulsed-liquid fast cycles according to themanufacturer's instructions (Applied Biosystems, California, USA).

[0128] For direct N-terminal sequencing, the purified protein was passedthrough a Brownlee C2 Aquapore column (0.46×3 cm, 7 μm, AppliedBiosystems, California, USA) using the same solvent system as above.N-terminal sequencing was then performed as described above. As theprotein was not derivatized prior to sequencing, cysteine residues couldnot be determined.

[0129] The following peptide sequences were found: N-terminal:Ser-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu- (SEQ ID NO:1)Leu-Gln-Leu-Phe-Ala-Gln-Trp-Ser-Ala- Ala-Ala-Tyr-X-Ser-Asn-Asn Internalpeptide 1: Val-His-Thr-Gly-Phe-Trp-Lys (SEQ ID NO:2) Internal peptide 2:Ala-Trp-Glu-Ser-Ala-Ala-Asp-Glu-Leu- (SEQ ID NO:3) Thr-Ser-Lys-Ile-Lys

[0130] No further peptides could be purified from the HPLC fractionationpresumably because they were very hydrophobic and therefore tightlybound to the reverse phase column.

[0131] A search in SWISS-PROT database release 31 for amino acidsequences with homology to the above peptides was performed and onlythree sequences were found.

[0132] All of the above peptides showed a low homology to the aboveknown sequences. Especially internal peptide 2 has very low homology tothe three lipases, LIP-RHIDL, LIP-RHIMI and MDLA-PENCA from Rhizopusdelamar (Haas and Berka, Gene, 1991, 109:107-113), Rhizomucor miehei(Boel et al., Lipids, 1988, 23:701-706) and Penicillium camenbertii(Yamaguchi et al., Gene, 1991, 103:61-67; Isobe and Nokihara, Febs.Lett., 1993, 320:101-106) respectively. Although the homology was notvery high it was possible to position the lipase 3 peptides on thesesequences as it is shown in the below Table 3.1. TABLE 3.1 Alignment oflipase 3 peptides with known lipase sequences LIP_RHIDLMVSFISISQGVSLCLLVSSMMLGSSAVPVSGKSGSSNTAVSASDNAALPP 50 LIP_RHIMIMVLKQRANYLGFLIVFFTAFLV--EAVPIKRQSNSTVDS--------LPP 40 MDLA_PENCAMRLS-----------FFTAL------------------SAVASLGYALPG 21*              . ...                   .        ** N-Terminal           SVSTSTLDELQLFAQWSAAAYXSNN LIP_RHIDLLISSRCAPPSNKGSKSDLQAEPYNMQKNTEWYESHGGNLTSIGKRDDNLV 100 LIP_RHIMILIPSRTSAPSSSPSTTDPEAPAM----------SRNGPLPS----DVETK 76 MDLA_PENCAKLQSR------DVSTSELDQFEFWVQYAAASY------------------ 47 . **      . *... .. LIP_RHIDLGGMTLDLPSDAPPISLSSSTNSASDGGKVVAATTAQIQEFTKYAGIAATA 150 LIP_RHIMIYGMALNATSYPDSV-----VQAMSIDGGIRAATSQEINELTYYTTLSANS 121 MDLA_PENCA-------------------------------------YEADYTAQVGDKL 60                                      * .  . .... LIP_RHIDLYCRSVVPGNKWDCVQCQKWVPDGKIITTFT-SLLSDTNGYVLRSDKQKTI 199 LIP_RHIMIYCRTVIPGATWDCIHCDA-TEDLKIIKTWS-TLIYDTNAMVARGDSEKTI 169 MDLA_PENCASCSKG------NCPEVEA--TGATVSYDFSDSTITDTAGYIAVDHTNSAV 102 *..       .* . .    . ..  ... . . **.. .  ....... Peptide 1                            VHTGFWK Peptide 2                                      AWESAADELTSK LIP_RHIDLYLVFRGTNSFRSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPV 249 LIP_RHIMIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVAT 219 MDLA_PENCAVLAFRGSYSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKE 152 ..***. * *. ..* .*   .    .*  .. ** ...  . .. Peptide 2 IK LIP_RHIDLVQEQLTAHPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVG 299 LIP_RHIMIVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSSNLFLYTQG 269 MDLA_PENCALKEVVAQNPNYELVVVGHSLGAAVATLAATDL--RGKGYPSAKLYAYA-- 198. .   . *.*.. *.*****.* * * . **  *.   .. .*  .. LIP_RHIDLGPRVGNPTFAYYVESTGIPFQRTVHKRDIVPHVPPQSFGFLHPGVESWIK 349 LIP_RHIMIQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWIT 319 MDLA_PENCASPRVGNAALAKYITAQGNNF-RFTHTNDPVPKLPLLSMGYVHVSPEYWIT 247 ****....* *. . *  . * ....* **..*  ..*..* . * **. LIP_RHIDLSGTSN-V-----QICTSEIETKDCSNSIVPFTSILD-HLSYF-DINEGSC 391 LIP_RHIMIDNSPETV-----QVCTSDLETSDCSNSIVPFTSVLD-HLSYF-GINTGLC 362 MDLA_PENCASPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDAGKG 297. . . *     .. ..... .. ... .*. . .. *. **  ...* LIP_RHIDL -------L  392LIP_RHIMI -------T  363 MDLA_PENCA PGLPFKRV  305

EXAMPLE 4 Isolation and Purification of Aspergillus tubigensis GenomicDNA

[0133] The Aspergillus tubigensis mutant strain was grown in PDB (Difco)for 72 hours and the mycelium was harvested. 0.5-1 g of mycelium wasfrozen in liquid nitrogen and ground in a mortar. Following evaporationof the nitrogen, the ground mycelium was mixed with 15 ml of anextraction buffer (100 mM Tris-HCl, pH 8.0, 50 mM EDTA, 500 mM NaCl, 10mM β-mercaptoethanol) and 1 ml 20% sodium dodecylsulfate. The mixturewas vigorously mixed and incubated at 65° C. for 10 min. 5 ml 3Mpotassium acetate, (pH 5.1 adjusted with glacial acetic acid) was addedand the mixture further incubated on ice for 20 min. The cellular debriswas removed by centrifugation for 20 min. at 20,000×g and 10 mlisopropanol was added to the supernatant to precipitate (30 min at −20°C.) the extracted DNA. After further centrifugation for 15 min at20,000×g, the DNA pellet was dissolved in 1 ml TE (10 mM Tris.HCl pH8.0, 1 mM EDTA) and precipitated again by addition of 0.1 ml 3 M NaAc,pH 4.8 and 2.5 ml ethanol. After centrifugation for 15 min at 20,000×gthe DNA pellet was washed with 1 ml 70% ethanol and dried under vacuum.Finally, the DNA was dissolved in 200 μl TE and stored at −20° C.

EXAMPLE 5 The Generation of a Fragment of the Putative Gene Coding forLipase 3 Using PCR

[0134] To obtain a fragment of the putative gene (in the followingreferred to as the lipA gene) as a tag to isolate the complete gene, aPCR amplification procedure based on the information in the isolatedpeptide sequences was carried out.

[0135] Degenerated primers for PCR amplification of a fragment of thelipase gene were designed based on the amino acid sequences of theisolated peptides. The following three PCR primers were synthesised:C035: TTC CAR AAN CCN GTR TGN AC (SEQ ID NO:4) 20 mer 256 mixture, basedon peptide 1 sequence VHTGFWK (Reversed). C036: CAR YTN TTY GCN CAR TGG(SEQ ID NO:5) 18 mer 256 mixture, based on the N-terminal sequenceQLFAQW C037: GCV GCH SWY TCC CAV GC (SEQ ID NO:6)

[0136] 17 mer 216 mixture, based on internal peptide 2 sequence AWESAA(reversed).

[0137] The oligonucleotides were synthesised on a Applied Biosystemsmodel 392 DNA/RNA Synthesizer. To reduce the degree of degeneracy therare Ala codon GCA and the Ser codon TCA have been excluded in design ofprimer C037.

[0138] With these primers the desired fragments were amplified by PCR.Using these primers it was expected that a fragment of about 300 bpshould be amplified provided there are no introns in the fragment.

[0139] The following PCR reactions were set up in 0.5 ml PCR tubes toamplify a putative lipA fragment:

[0140] 1. 0.5 μg total genomic DNA, 100 pmol primer C036, 100 pmolprimer C037, 10 μl PCR Buffer II (Perkin Elmer), 6 μl 25 mM MgCl₂, 2 μldNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTT 2 units Amplitaqpolymerase (Perkin Elmer), and water to a total volume of 100 μl.

[0141] 2. 0.5 μg total genomic DNA, 100 pmol primer C035, 100 pmolprimer C036, 10 μl PCR Buffer II (Perkin Elmer), 6 μl 25 mM MgCl₂, 2 μldNTP mix (10 mM DATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTT 2 units Amplitaqpolymerase (Perkin Elmer), and water to a total volume of 100 μl.

[0142] The reactions were performed using the following program: 94° C.2 min 94° C. 1 min ) 40° C. 1 min ) 72° C. 1 min ) These three stepswere repeated for 30 72° C. 5 min cycles  5° C. SOAK

[0143] The PCR amplifications were performed in a MJ Research Inc.PTC-100 Thermocycler.

[0144] In reaction 1, three distinct bands of about 300, 360 and 400 bp,respectively could be detected. These bands were isolated and clonedusing the pT7-Blue-T-vector kit (Novagene). The sizes of these fragmentis in agreement with the expected size provided that the fragmentcontains 0, 1 or 2 introns, respectively.

[0145] The three fragments were sequenced using a “Thermo Sekvenasefluorescent labelled primer cycle sequencing Kit” (Amersham) andanalyzed on a ALF sequencer (Pharmacia) according to the instructions ofthe manufacturer. The fragment of about 360 bp contained a sequence thatwas identified as a lipase and, as it contained the part of theN-terminal distal to the sequence used for primer design, it wasconcluded that the desired lipA gene fragment was obtained.

[0146] The sequence of the about 360 bp PCR fragment (SEQ ID NO:7) isshown in the following Table 5.1. The peptide sequence used for primerdesign is underlined. The remaining part of the N-terminal sequence isdoubly underlined. TABLE 5.1 PCR-generated putative lipA sequence        10        20        30        40        50        60         |         |         |         |         |         |tacccggggntccgattCAGTTGTTCGCGCAATGGTCTGCCGCAGCTTATTGCTCGAATA                  Q  L  F  A  Q  W  S  A  A  A  Y  C  S  N        70        80        90       100       110       120         |         |         |         |         |         |ATATCGACTCGAAAGAVTCCAACTTGACATGCACGGCCAACGCCTGTCCATCAGTCGAGG N  I  D  S  K  X  S  N  L  T  C  T  A  N  A  C  P  S  V  E       130       140       150       160       170       180         |         |         |         |         |         |AGGCCAGTACCACGATGCTGCTGGAGTTCGACCTGTATGTCACTCAGATCGCAGACATAGE  A  S  T  T  M  L  L  E  F  D  L  Y  V  T  Q  I  A  D  I       190       200       210       220       230       240         |         |         |         |         |         |AGCACAGCTAATTGAACAGGACGAACGACTTTTGGAGGCACAGCCGGTTTCCTGGCCGCGE  H  S  -  L  N  R  T  N  D  F  W  R  H  S  R  F  P  G  R       250       260       270       280       290       300         |         |         |         |         |         |GACAACACCAACAAGCGGCTCGTGGTCGCCTTCCGGGGAAGCAGCACGATTGAGAACTGGG  Q  H  Q  Q  A  A  R  G  R  L  P  G  K  Q  H  D  -  E  L       310       320       330          |         |         |ATTGCTAATCYTGACTTCATCCTGGRAGATAACG D  C  -  X  -  L  H  P  X  R  -

[0147] The finding of this sequence permitted full identification of thePCR fragment as part of the lipA gene. The stop codon found in thereading frame can be caused either by a PCR or a reading error or therecan be an intron encoded in the fragment as a consensus intron start andending signal (shown in bold). If the putative intron is removed a shiftin reading frame will occur. However, an alignment of the deduced aminoacid sequence and the fungal lipases shown in Table 3.1 suggested thatthe fragment was part of the desired gene.

EXAMPLE 6 Cloning and Characterisation of the LipA Gene

[0148] (i) Construction of an Aspergillus tubigensis genomic library

[0149]Aspergillus tubigensis genomic DNA was digested partially withTsp5091 (New England Biolabs Inc.). 10 μg DNA was digested in 100 μlreaction mixture containing 2 units Tsp5091. After 5, 10, 15 and 20minutes 25 μl was removed from the reaction mixture and the digestionwas stopped by addition of 1 μl 0.5 M EDTA, pH 8.0. After all fourreactions had been stopped, the samples were run on a 1% agarose gel inTAE buffer (10×TAE stock containing per litre: 48.4 g Trizma base, 11.5ml glacial acetic acid, 20 ml 0.5 M EDTA pH 8.0). HindIII-digested phageLambda DNA was used as molecular weight marker (DNA molecular weightmarker II, Boehringer, Mannheim). Fragments of a size between about 5and 10 kb were cut out of the gel and the DNA fragments were purifiedusing Gene Clean II Kit (Bio-101 Inc.). The purified fragments werepooled and 100 ng of the pooled fragments were ligated into 1 μgEcoRI-digested and dephosphorylated ZAP II vector (Stratagene) in atotal volume of 5 μl. 2 μl of this volume was packed with Gigapack IIpacking extract (Stratagene) which gave a primary library of 650,000pfu.

[0150]E. coli strain XL1-Blue-MRF (Stratagene) was infected with5×50,000 pfu of the primary library. The infected bacteria were mixedwith top agarose (as NZY plates but with 6 g agarose per litre insteadof the agar) and plated on 5 NZY plates (13 cm). After incubation at 37°C. for 7 hours, 10 ml SM buffer (per litre: 5.8 g NaCl, 2.0 gMgCl₂.7H₂O, 50 ml 1 M Tris.HCl pH 7.5, 5.0 ml of 2% (w/v) gelatine) andincubated overnight at room temperature with gently shaking. The buffercontaining washed-out phages was collected and pooled. 5% chloroform wasadded and after vigorous mixing the mixture was incubated 1 hour at roomtemperature. After centrifugation for 2 minutes at 10,000×g the upperphase containing the amplified library was collected anddimethylsulphoxide was added to 7%. Aliquots of the library was takenout in small tubes and frozen at −80° C. The frozen library contained2.7×10⁹ pfu/ml with about 6% without inserts.

[0151] (ii) Screening of the Aspergillus tubigensis library

[0152] 2×50.000 pfu were plated on large (22×22 cm) NZY platescontaining a medium containing per litre: 5 g NaCl, 2 g MgSO₄.7H₂O, 5 gyeast extract, 10 g casein hydrolysate, 15 g agar, pH adjusted to 7.5with NaOH. The medium was autoclaved and cooled to about 60° C. andpoured into the plates. Per plate was used 240 ml of medium.

[0153] The inoculated NZY plates were incubated overnight at 37° C. andplaque lifts of the plates were made. Two lifts were made for each plateon Hybond N (Amersham) filters. The DNA was fixed using UV radiation for3 min. and the filters were hybridized as described in the followingusing, as the probe, the above PCR fragment of about 360 bp that waslabelled with ³²P-dCTP using Ready-to-Go labelling kit (Pharmacia).

[0154] The filters were prehybridised for one hour at 65° C. in 25 mlprehybridisation buffer containing 6.25 ml 20×SSC (0.3 M Na₃citrate, 3 MNaCl), 1,25 ml 100×Denhard solution, 1.25 ml 10% SDS and 16.25 ml water.150 μl 10 mg/ml denatured Salmon sperm DNA was added to theprehybridization buffer immediately before use. Followingprehybridization, the prehybridisation buffer was discarded and thefilters hybridised overnight at 65° C. in 25 ml prehybridisation bufferwith the radiolabelled PCR fragment.

[0155] Next day the filters were washed according to the followingprocedure: 2×15 min. with 2×SSC+0.1% SDS, 15 min. with 1×SSC+0.1% SDSand 10 min. with 0.1×SSC+0.1% SDS.

[0156] All washes were done at 65° C. The sheets were autoradiographedfor 16 hours and positive clones were isolated. A clone was reckoned aspositive only if there was a hybridisation signal on both plaque liftsof the plate in question.

[0157] Seven putative clones were isolated and four were purified byplating on small petri dishes and performing plaque lifts essentially asdescribed above.

[0158] The purified clones were converted to plasmids using an ExAssistKit (Stratagene).

[0159] Two sequencing primers were designed based on the about 360 bpPCR fragment. The sequencing primers were used to sequence the clonesand a positive clone with the lipA gene encoding lipase 3 was found. Theisolated positive clone was designated pLIP4.

[0160] (iii) Characterisation of the pLIP4 clone

[0161] A restriction map of the clone was made. The above 360 bp PCRfragment contained a SacII site and as this site could be found in thegenomic clone as well this site facilitated the construction of the map.The restriction map showing the structure of pLIP4 is shown in FIG. 1.The restriction map shows that the complete gene is present in theclone. Additionally, since promoter and terminator sequences arepresent, it was assumed that all the important regions is present in theclone.

[0162] A sample of Escherichia coli strain DH5α containing pLIP4 wasdeposited in accordance with the Budapest Treaty with The NationalCollections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St.Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 lRY on Feb. 24,1997 under the accession number NCIMB 40863.

[0163] The gene was sequenced using cycle sequencing and conventionalsequencing technology. The complete sequence (SEQ ID NO:8) is shownbelow in Table 6.1. The sequence has been determined for both strandsfor the complete coding region and about 100 bp upstream and downstreamof the coding region. The sequences downstream to the coding region haveonly been determined on one strand and contains a few uncertainties. Inthe sequence as shown below, the intron sequences are indicated aslowercase letters and the N-terminal and the two internal peptides(peptide 1 and peptide 2) are underlined: TABLE 6.1 The DNA sequence forthe lipA gene and flanking sequences 1CCNDTTAATCCCCCACCGGGGTTCCCGCTCCCGGATGGAGATGGGGCCAAAACTGGCAAC 61CCCCAGTTGCGCAACGGAACAACCGCCGACCCGGAACAAAGGATGCGGATGAGGAGATAC 121GGTGCCTGATTGCATGGCTGGCTTCATCTGCTATCGTGACAGTGCTCTTTGGGTGAATAT 181TGTTGTCTGACTTACCCCGCTTCTTGCTTTTTCCCCCCTGAGGCCCTGATGGGGAATCGC 241GGTGGGTAATATGATATGGGTATAAAAGGGAGATCGGAGGTGCAGTTGGATTGAGGCAGT 301GTGTGTGTGTGCATTGCAGAAGCCCGTTGGTCGCAAGGTTTTGGTCGCCTCGATTGTTTG 361TATACCGCAAGATGTTCTCTGGACGGTTTGGAGTGCTTTTGACAGCGCTTGCTGCGCTGG            M  F  S  G  R  F  G  V  L  L  T  A  L  A  A  L 421GTGCTGCCGCGCCGGCACCGCTTGCTGTGCGGAgtaggtgtgcccgatgtgagatggttgG  A  A  A  P  A  P  L  A  V  R 481gatagcactgatgaagggtgaatagGTGTCTCGACTTCCACGTTGGATGAGTTGCAATTG                         S  V  S  T  S  T  L  D  E  L  Q  L 541TTCGCGCAATGGTCTGCCGCAGCTTATTGCTCGAATAATATCGACTCGAAAGACTCCAAC F  A  Q  W  S  A  A  A  Y  C  S  N  N  I  D  S  K  D  S  N 601TTGACATGCACGGCCAACGCCTGTCCATCAGTCGAGGAGGCCAGTACCACGATGCTGCTG L  T  C  T  A  N  A  C  P  S  V  E  E  A  S  T  T  M  L  L 661GAGTTCGACCTgtatgtcactcagatcgcagacatagagcacagctaatttgaacagGAC E  F  D  L                                                T 721GAACGACTTTGGAGGCACAGCCGGTTTCCTGGCCGCGGACAACACCAACAAGCGGCTCGT  N  D  F  G  G  T  A  G  F  L  A  A  D  N  T  N  K  R  L  V 781GGTCGCCTTCCGGGGAAGCAGCACGATTGAGAACTGGATTGCTAATCTTGACTTCATCCT  V  A  F  R  G  S  S  T  I  E  N  W  I  A  N  L  D  F  I  L 841GGAAGATAACGACGACCTCTGCACCGGCTGCAAGGTCCATACTGGTTTCTGGAAGGCATG  E  D  N  D  D  L  C  T  G  C  K  V  H  T  G  F  W  K  A  W 901GGAGTCCGCTGCCGACGAACTGACGAGCAAGATCAAGTCTGCGATGAGCACGTATTCGGG  E  S  A  A  D  E  L  T  S  K  I  K  S  A  M  S  T  Y  S  G 961CTATACCCTATACTTCACCGGGCACAGTTTGGGCGGCGCATTGGCTACGCTGGGAGCGAC  Y  T  L  Y  F  T  G  H  S  L  G  G  A  L  A  T  L  G  A  T 1021AGTTCTGCGAAATGACGGATATAGCGTTGAGCTGgtgagtccttcacaaaggtgatggag  V  L  R  N  D  G  Y  S  V  E  L 1081cgacaatcgggaacagacagtcaatagTACACCTATGGATGTCCTCGAATCGGAAACTAT                            Y  T  Y  G  C  P  R  I  G  N  Y 1141GCGCTGGCTGAGCATATCACCAGTCAGGGATCTGGGGCCAACTTCCGTGTTACACACTTG A  L  A  E  H  I  T  S  Q  G  S  G  A  N  F  R  V  T  H  L 1201AACGACATCGTCCCCCGGGTGCCACCCATGGACTTTGGATTCAGTCAGCCAAGTCCGGAA N  D  I  V  P  R  V  P  P  M  D  F  G  F  S  Q  P  S  P  E 1261TACTGGATCACCAGTGGCAATGGAGCCAGTGTCACGGCGTCGGATATCGAAGTCATCGAG Y  W  I  T  S  G  N  G  A  S  V  T  A  S  D  I  E  V  I  E 1321GGAATCAATTCAACGGCGGGAAATGCAGGCGAAGCAACGGTGAGCGTTGTGGCTCACTTG G  I  N  S  T  A  G  N  A  G  E  A  T  V  S  V  V  A  H  L 1381TGGTACTTTTTTGCGATTTCCGAGTGCCTGCTATAACTAGACCGACTGTCAGATTAGTGG W  Y  F  F  A  I  S  E  C  L  L  - 1441ACGGGAGAAGTGTACATAAGTAATTAGTATATAATCAGAGCAACCCAGTGGTGGTGATGG 1501TGGTGAAAGAAGAAACACATTGAGTTCCCATTACGKAGCAGWTAAAGCACKTKKGGAGGC 1561GCTGGTTCCTCCACTTGGCAGTTGGCGGCCATCAATCATCTTTCCTCTCCTTACTTTCGT 1621CCACCACAACTCCCATCCTGCCAGCTGTCGCATCCCCGGGTTGCAACAACTATCGCCTCC 1681GGGGCCTCCGTGGTTCTCCTATATTATTCCATCCGACGGCCGACGTTTCACCCTCAACCT 1741GCGCCGCCGCAAAATCTCCCCGAGTCGGTCAACTCCCTCGAACCGCCGCCCGCATCGACC 1801TCACGACCCCGACCGTCTGYGATYGTCCAACCG

[0164] (iv) Analysis of the sequence of the complete gene

[0165] The peptide sequences obtained could all be found in the deducedamino acid sequence (see Table 5.1) which confirms again that thesequence found is the sequence of the lipase 3 gene. The gene wasdesignated lipA.

[0166] The amino acid sequence was aligned with the three fungal lipasesused to align the peptide sequences. The alignment is shown in Table6.2. TABLE 6.2 Alignment of the lipase 3 sequence with known fungallipases LIPASE3 MFSG-----------RFGVLL------------------------TALAA 15MDLA_PENCA MRLS-----------FFTAL-------------------------SAVAS 14LIP_RHIDL MVSFISISQGVSLCLLVSSMMLGSSAVPVSGKSGSSNTAVSASDNAALPP 50LIP_RHIMI MVLKQRANYLGFLIVFFTAFLV--EAVPIKRQSNSTVDS--------LPP 40*                . .                           ... LIPASE3L------------------------------------------------- 16 MDLA_PENCAL------------------------------------------------- 15 LIP_RHIDLLISSRCAPPSNKGSKSDLQAEPYNMQKNTEWYESHGGNLTSIGKRDDNLV 100 LIP_RHIMILIPSRTSAPSSSPSTTDPEAPAM----------SRNGPLPS----DVETK 76 * LIPASE3--------GAAAPAPLA-----------VRSVSTSTLDELQLFAQWSAAA 47 MDLA_PENCA--------GYALPGKLQ-----------SRDVSTSELDQFEFWVQYAAAS 46 LIP_RHIDLGGMTLDLPSDAPPISLSSSTNSASDGGKVVAATTAQIQEFTKYAGIAATA 150 LIP_RHIMIYGMALNATSYPDSV-----VQAMSIDGGIRAATSQEINELTYYTTLSANS 121        . . .                  ....  ....  .. .*.. LIPASE3YCSNNIDSK-DSNLTCTANACPSVEEASTTMLLEFDLTNDFGGTAGFLAA 96 MDLA_PENCAYYEADYTAQVGDKLSCSKGNCPEVEATGATVSYDFS-DSTITDTAGYIAV 95 LIP_RHIDLYCRSVVP---GNKWDCVQ--CQKWVPDGKIIT---TFTSLLSDTNGYVLR 192 LIP_RHIMIYCRTVIP---GATWDCIH--CDA-TEOLKIIK---TWSTLIYDTNAMVAR 162*. .       ... .*    *      .  ..    . .. . .*.. . LIPASE3DNTNKRLVVAFRGSSTIENWIANLDFILEDNDDLCTGCKVHTGFWKAWES 146 MDLA_PENCADHTNSAVVLAFRGSYSVRNWVADATFV-HTNPGLCDGCLAELGFWSSWKL 144 LIP_RHIDLSDKQKTIYLVFRGTNSFRSAITDIVFNFSDYKPV-KGAKVHAGFLSSYEQ 241 LIP_RHIMIGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPV-SGTKVHKGFLDSYGE 211..... . ..***. .  . ...  *   .   . .*  .. ** ... LIPASE3AADELTSKIKSAMSTYSGYTLYFTGHSLGGALATLGATVL--RNDGY-SV 193 MDLA_PENCAVRDDIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDL--RGKGYPSA 192 LIP_RHIDLVVNDYFPVVQEQLTAHPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPK 291 LIP_RHIMIVQNELVATVLDQFKQYPSYKVAVTGHSLGGATALLCALDLYQREEGLSSS 261. ..     . .     ..*..  .*****.* * * .  *  *.   . LIPASE3ELYTY--GCPRIGNYALAEHITSQGSGANFRVTHLNDIVPRVPPMDFGFS 241 MDLA_PENCAKLYAY--ASPRVGNAALAKYITAQGN--NFRFTHTNDPVPKLPLLSMGYV 238 LIP_RHIDLNLSIFTVGGPRVGNPTFAYYVESTGIPFQ-RTVHKRDIVPHVPPQSFGFL 340 LIP_RHIMINLFLYTQGQPRVGDPAFANYVVSTGIPYR-RTVNERDIVPHLPPAAFGFL 310.*  .  . **.*. ..* .. . *   . * .. .* **..*  ..*. LIPASE3QPSPEYWITSGNGASVTASDIEVIEGINSTAGNAGEATVSVV---AHLWY 288 MDLA_PENCAHVSPEYWITSPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWY 288 LIP_RHIDLHPGVESWIKSGTSN-VQICTSEIE------TKDCSNSIVPFTSILDHLSY 383 LIP_RHIMIHAGEEYWITDNSPETVQVCTSDLE------TSDCSNSIVPFTSVLDHLSY 354. . * **.. . *  .. ..        . . ...    .    .*. * LIPASE3FFAISECL--------L  297 MDLA_PENCA FVQVDAGKGPGLPFKRV  305 LIP_RHIDLF-DINEGSC-------L  392 LIP_RHIMI F-GINTGLC-------T  363 *  ...

[0167] The above alignment shows that lipase 3 is homologous to theknown lipase sequences but that the homology is not very high. Deletionsor insertions in the lipase 3 sequence was not observed when comparingthe sequence with these three lipases. This strengthens the probabilitythat the putative introns have been identified correctly.

[0168] A search in SWISS-PROT release 31 database was performed and itdid not lead to further sequences with higher homology than that to theabove known lipases (Table 6.3).

[0169] The sequence with highest homology is a mono-diacyl lipase fromPenicillium camembertii where the identity is found to 42%. However theC-terminal of lipase 3 resembles the 2 lipases from Zygomycetes(Rhizopus and Rhizomucor) and not the P. camembertii enzyme. TABLE 6.3Alignment of coding sequences of the lipA gene and gene coding formono-diacyl lipase from Penicillium camemberti LIPASE3   -MFSGRFGVLLTALAALGAAAPAPLAVRSVSTSTLDELQLFAQWSAAAYCS -50|    |  |   | || | |  |  | |||| ||       |  || | MDLA_PENCA-MRLSFFTAL-SAVASLGYALPGKLQSRDVSTSELDQFEFWVQYAAASYYE -49 LIPASE3   -NNIDSK-DSNLTCTANACPSVEEASTTMLLEFDLTNDFGGTAGFLAADNT -99          | |    || ||    |    |        |||  | | | MDLA PENCA-ADYTAQVGDKLSCSKGNCPEVEATCATVSYDFS-DSTITDTACYIAVDHT -98 LIPASE3   -NKRLVVAFRGSSTIENWIANLDFILEDNDDLCTGCKVHTGFWKAWESAAD -149|   | |||||    || |   |    |  || ||    |||  |    | MDLA_PENCA-NSAVVLAFRGSYSVRNWVADATFV-HTNPGLCDGCLAELGFWSSWKLVRD -147 LIPASE3   -ELTSKIKSAD4STYSGYTLYFTGHSLGGALATLGATVLRNDGY-SVELYTY -198      |        | |   ||||| | ||| || ||  || |  || | MDLA_PENCA-DIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAY -197 LIPASE3   -GCPRIGNYALAEHITSQGSGANFRVTHLNDIVPRVPPMDFGFSQPSPEYW -248  || || |||  || ||   ||| || ||  |    |     ||||| MDLA_PENCA-ASPRVGNAALAKYITAQGN--NFRFTBTNDPVPKLPLLSMGYVHVSPEYW -245 LIPASE3   -ITSGNGASVTASDIEVIEGINSTAGNAGEATVSVV---AHLWYFFAISEC -295||| | | |  ||| || |  |  || |          || ||| MDLA PENCA-ITSPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDAG -295 LIPASE3   -L--------L  -297 MDLA_PENCA- KGPGLPFKRV  -305

[0170] The N-terminal of the mature lipase has been determined byN-terminal sequencing to be the serine residue No. 28 of the lipase 3precursor (SEQ ID NO:9) as shown in Table 6.4 below. Hence the aminoacids No. 1 to No. 27 is the signal sequence. TABLE 6.4 Amino acidsequence of the precursor of lipase 3         5       10        15        20        25        30         |        |         |         |         |         | 1 M F S G RF G V L L T A L A A L G A A A P A P L A V R S V S 31 T S T L D E L 0 L FA Q W S A A A Y c S N N I D S K D S N L 61 T C T A N A C P S V E E A S TT M L L E F D L T N D F G G T 91 A G F L A A D N T N K R L V V A F R G SS T I E N W I A N L 121 D F I L E D N D D L C T G C K V H T G F W K A WE S A A D E 151 L T S K I K S A M S T Y S G Y T L Y F T G H S L G G A LA T 181 L G A T V L R N D G Y S V E L Y T Y G C P R I G N Y A L A E 211H I T S Q G S G A N F R V T H L N D I V P R V P P M D F G F 241 S Q P SP E Y W I T S G N G A S V T A S D I E V I E G I N S 271 T A G N A G E AT V S V V A H L W Y F F A I S E C L L

[0171] Residues 167-176 are recognised as a common motif for the serinelipases (PROSITE). The crystal structure for the Rhizomucor mieheiserine lipase has been examined and the residues in the active siteidentified (Brady et al., Nature, 1990, 343:767-770; Derewanda et al.,J. Mol. Biol., 1992, 227:818-839). The active site residues of R. mieheilipase have all been conserved in all the lipases and correspond to thefollowing residues in lipase 3: serine 173, aspartic acid 228 andhistidine 285.

[0172] Lipase 3 contains 7 cysteine residues. Four of these areconserved in the P. camembertii lipase where they form disulphide bonds(Isobe and Nokuhara, Gene, 1991, 103:61-67). This corresponds todisulphide bonds between residue 62-67 and 131-134. In addition, twocysteine residues are homologous to two C residues which forms anadditional disulphide bond in Rhizopus and Rhizomucor lipasescorresponding to residues 49-295.

[0173] Two putative N-glycosylation sites were found in lipase 3 inposition 59 and 269. Neither of these are conserved in the other fungallipases.

EXAMPLE 7 Transformation of Aspergillus tubigensis and Overexpression ofLipase 3 in A. tubigensis

[0174] The protocol for transformation was based on the teachings ofBuxton et al. (Gene, 1985, 37:207-214), Daboussi et al (Curr. Genet.,1989, 15:453-456) and Punt and van den Hondel, (Meth. Enzym., 1992,216:447-457).

[0175] A multicopy lipA strain was produced by transforming the pLIP4plasmid into Aspergillus tubigensis strain 6M 179 using cotransformationwith a hygromycin resistant marker plasmid.

[0176] A screening procedure used to visualise fungal lipase afterultrathin layer isoelectric focusing was adapted to screen Aspergillustubigensis transformants grown on agar plates. Screening of lipaseproducers on agar plates was done using 2% olive oil as the substratefor the enzyme (lipase) as well as the inducer for the lipase promoter.In addition, the plates contained a fluorescent dye, Rhodamine B. In thepresence of olive oil, the transformants will be induced to secretelipase. The lipase secreted into the agar plate will hydrolyse the oliveoil causing the formation of orange fluorescent colonies that is visibleupon UV radiation (350 nm). The appearence of fluorescent colonies wasgenerally monitored after 24 hours of growth. After several days ofgrowth, the lipase producing strains could be identified as orangefluorescent strains that are visible by eye. Under this plate screeningcondition, the untransformed strain gave no background fluorescence andappeared as opaque pink colonies.

[0177] Sixteen transformants that showed orange fluorescent halos werecultivated for 8 days in shake flasks containing 100 ml of minimalmedium supplemented with 1% olive oil, 0.5% yeast extract and 0.2%casamino acids. The amount of lipase secreted was quantified by applying10 μl of cell-free culture supernatant into holes punched in oliveoil—Rhodamine B agar plates and incubating the plates overnight at 37°C. Five transformants with higher lipase production were found.

[0178] The cell-free culture supernatants from the five transformantswere desalted using NAP 5 columns (Pharmacia) and equilibrated in 1Mammonium sulfate (50 mM sodium acetate, pH 5.5). The desalted culturesupernatants were fractionated by hydrophobic interaction chromatography(HIC) on a Biogel Phenyl-5 PW column (Biorad). Elution was done by adescending salt gradient of 1M to 0 M ammonium sulfate (20 mM sodiumacetate, pH 5.5). A single discrete protein peak was observed afterfractionation. The area of the protein peaks were calculated among thedifferent transformants and compared with the untransformed strain. Thebest transformant showed a 62-fold increase in the amount of lipaseafter HIC fractionation. A chromatogram of the HIC fractionated culturesupernatant of this transformant is shown in FIG. 3 and a similarchromatogram for the untransformed strain is shown in FIG. 4.

[0179] The fraction containing the transformed lipase was freeze-dried.The transformed lipase was carboxymethylated and subjected to N-terminalamino acid sequencing of the first 15 amino acids and it was found thatthe sequence of the recombinant lipase was exactly the same as thenative lipase indicating correct signal sequence cleavage.

[0180] The different lipase fractions collected after HIC were separatedon a 12% Tris-Glycine SDS gel and silver staining revealed one proteinband, confirming the homogeneity of the fractions. In addition, thecrude extract showed a major lipase band as the only band thataccumulated in the culture supernatant in very high amounts when thefungus was cultured in the olive oil-containing medium.

[0181] The recombinant lipase was analysed by matrix-assisted laserdesorption ionisation (MALDI) by means of a time-of-flight (TOF) massspectrometer as described hereinbefore. The molecular weight of therecombinant lipase was 32,237 Da.

[0182] Detection of N-linked oligosaccharides was achieved by digestionof the lipase with endo-β-N-acetyl-glucosamidase H from Streptomyces(Sigma). Digestion of recombinant lipase secreted into the growth mediumaltered the mobility of the band seen on SDS-PAGE which moved as asingle band with a olecular mass of about 30 kDa.

[0183] Deglycosylated recombinant lipase generated by digestion withndoglycosidase and analysed directly by MALDI mass spectroetry gave amolecular weight of the polypeptide backbone of 9,325 Da.

[0184] C. Baking Experiments

EXAMPLE 8 Baking Experiments Using Lipase 3

[0185]8.1. Baking procedures and analytical methods

[0186] (i) Baking procedure for Danish toast bread

[0187] Flour (Danish reform flour) 2000 g, dry yeast 30 g, salt 30 g andwater corresponding to 400 Brabender units +3%, was kneaded in a HobartMixer with hook for 2 min. at low speed and 10 min. at high speed. Doughtemperature after kneading was 25° C. Resting time was 10 min. at 30° C.The dough was scaled 750 g per dough and rested again for 5 min at 33°C. and 85% RH. After moulding on a Glimik moulder, the dough wereproofed in tins for 50 min at 33° C. and baked in a Wachtel oven for 40min at 22° C. with steam injection for 16 sec. After cooling, the breadwas scaled and the volume of the bread was measured by the rape seeddisplacement method. The specific volume is calculated by dividing thebread volume (ml) by the weight (g) of the bread.

[0188] The crumb was evaluated subjectively using a scale from 1 to 5where 1=coarsely inhomogeneous and 5=nicely homogeneous.

[0189] Three breads baked in tins with lid were stored at 20° C. andused for firmness measurements and pore measurements by means of anImage Analyzer.

[0190] (ii) Baking procedure for Danish rolls

[0191] Flour (Danish reform) 1500 g, compressed yeast 90 g, sugar 24 g,salt 24 g and water corresponding to 400 Brabender units −2% werekneaded in a Hobart mixer with hook for 2 min. at low speed and 9 min athigh speed. After kneading, the dough temperature was 26° C. The doughwas scaled 1350 g. After resting for 10 min. at 30° C., the dough wasmoulded on a Fortuna moulder after which the dough was proofed for 45min. at 34° C. and baked in a Bago oven for 18 min. at 220° C. withsteam injection for 12 sec. After cooling, the rolls were scaled and thevolume of the rolls was measured by the rape seed displacement method.Specific volume is calculated as described above.

[0192] (iii) Determination of pore homogeneity

[0193] The pore homogeneity of the bread was measured by means of animage analyzer composed of a standard CCD-video camera, a videodigitiser and a personal computer with WinGrain software. For everybread, the results of pore diameter in mm and pore homogeneity werecalculated as an average of measurements from 10 slices of bread. Thepore homogeneity was expressed in % of pores that are larger than 0.5times the average of pore diameter and smaller than 2 times the averagediameter.

[0194] (iv) Determination of firmness

[0195] The firmness of bread, expressed as N/dm², was measured by meansof an Instron UTM model 4301 connected to a personal computer. Theconditions for measurement of bread firmness were: Load Cell Max. 100 NPiston diameter 50 mm Cross head speed 200 mm/min Compression 25%Thickness of bread slice 11 mm

[0196] The result was an average of measurements on 10 bread slices forevery bread.

[0197] (v) Determination of gluten index

[0198] Gluten index was measured by means of a Glutomatic 2200 fromPerten Instruments (Sweden). Immediately after proofing, 15 g of doughwas scaled and placed in the Glutomatic and washed with 500 ml 2% NaClsolution for 10 min. The washed dough was transferred to a Gluten IndexCentrifuge 2015 and the two gluten fractions were scaled and the glutenindex calculated according to the following equation:

Gluten index=(weight of gluten remaining on the sieve×100)/total-weightof gluten

[0199] (vi) Extraction of lipids from dough

[0200] 30 g of fully proofed dough was immediately frozen andfreeze-dried. The freeze-dried dough was milled in a coffee mill andpassed through a 235 μm screen. 4 g freeze-dried dough was scaled in a50 ml centrifuge tube with screw lid and 20 ml water saturated n-butanol(WSB) was added. The centrifuge tube was placed in a water bath at atemperature of 100° C. for 10 min. after which the tubes were placed ina Rotamix and turned at 45 rpm for 20 min. at ambient temperature. Thetubes were again placed in the water bath for 10 min. and turned on theRotamix for another 30 min. at ambient temperature.

[0201] The tubes were centrifuged at 10,000×g for 5 min. 10 ml of thesupernatant was pipetted into a vial and evaporated to dryness undernitrogen cover. This sample was used for HPLC analysis.

[0202] A similar sample was fractionated on a Bond Elut Si (Varian1211-3036). The non-polar fraction was eluted with 10 mlcyclohexan:isopropanol:acetic acid (55:45:1) and evaporated to dryness.This sample was used for GLC analysis.

[0203] (vii) HPLC analysis

[0204] Column: LiChrospher 100 DIOL 5 μm (Merck art. 16152) 250×4 mmwith a water jacket of a temperature of 50° C.

[0205] Mobile phases:

[0206] A: heptan:isopropanol:n-butanol:tetrahydrofuran:isooctan:water(64.5:17.5:7:5:5:1)

[0207] B: isopropanol:n-butanol:tetrahydrofuran:isooctan:water(73:7:5:5:10)

[0208] The mobile phases contained 1 mmol trifluoroacetic acid per 1mobile phase and were adjusted to pH 6.6 with ammonia.

[0209] Pump: Waters 510 equipped with a gradient controller.

[0210] Gradient: Flow ml/min Time (min) A (%) B (%) 1.0 0 100 0 1.0 25 0100 1.0 30 0 100 1.0 35 100 0 1.0 40 100 0

[0211] Detector: CUNOW DDL21 (evaporative light-scattering); temperature100° C.; voltage: 600 volt; air flow: 6.0 1/min.

[0212] Injector: Hewlett Packard 1050; injection volume: 50 μl.

[0213] The samples for analysis were dissolved in 5 mlchloroform:methanol (75:25), sonicated for 10 min and filtered through a0.45 μm filter.

[0214] (viii) GLC analysis

[0215] Perkin Elmer 8420 Capillary Gas Chromatograph equipped with WCOTfused silica column 12.5 m×0.25 mm coated with 0.1 μm stationary phaseof 5% phenyl-methyl-silicone (CP Sil 8 CB from Crompack).

[0216] Carrier: Helium

[0217] Injection: 1.5 μl with split

[0218] Detector: FID 385° C. Oven program: 1 2 3 4 Oven temperature, °C. 80 200 240 360 Isothermal time, min 2 0 0 10 Temperature rate, °C./min 20 10 12 —

[0219] Sample preparation: 50 mg non-polar fraction of wheat lipids wasdissolved in 12 ml heptane:pyridine (2:1) containing 2 mg/ml heptadecaneas internal standard. 500 μl of the solution was transferred to a crimpvial and 100 μl N-methyl-N-trimethylsilyl-trifluoracetamide was added.The mixture was allowed to react for 15 min at 90° C.

[0220] Calculation: Response factors for mono-, di- and triglyceridesand free fatty acids were determined from reference mixtures of thesecomponents. Based on these response factors, the glycerides and the freefatty acids were calculated in wheat lipids.

[0221] 8.2. Baking experiments with lipase 3 in Danish toast bread

[0222] The effect of adding lipase 3 to a dough for making Danish toastbread was evaluated. The enzyme was added as a freeze-dried preparationon maltodextrin together with the other ingredients. The results of thebaking tests are shown in Tables 8.1 to 8.4. TABLE 8.1 Lipase 0 5,000  15,000   25,000   LUS/kg flour Specific 4.43    4.43    4.22    4.37volume of bread Firmness 35   33   32   30 Day 1 Firmness 90   90   85  73 Day 7

[0223] TABLE 8.2 Lipase 0 5,000   15,000   25,000   LUS/kg flour Averagediameter of 2.96    2.33    2.47    2.65 the crumb pore, mm Homogeneityof 64.9    73.8    66.0    67.1 crumb pore, % Porosity, % 85.9    84.7   85.5    85.1 Gluten index, % 42    45.5   55   65

[0224] TABLE 8.3 Lipase 0 5,000   15,000   25,000   LUS/kg flour Fattyacids, % 0.090    0.148     0.218     0.241 Monoglycerides, % 0.017   0.031     0.035     0.039 Diglycerides, % 0.020    0.036     0.040    0.045 Triglycerides, % 0.790    0.714     0.673     0.622

[0225] TABLE 8.4 Lipase 0 5,000   15,000   25,000   LUS/kg flourMonogalactosyl 0.073    0.040    0.025    0.018 Diglyceride, %Digalactosyl 0.244    0.220    0.182    0.127 Diglyceride, %Digalactosyl 0.008    0.022    0.044    0.054 Monoglyceride, %Phosphatidyl 0.064    0.073    0.055    0.041 choline, %Lysophosphatidyl 0.164    0.182    0.171    0.165 choline, %

[0226] By the addition of up to about 5,000 LUS/kg flour of the lipaseno change in bread volume was observed, but at a higher dosage of lipase3 there was a tendency to a small but not statistically significantdecrease in volume (Table 8.1).

[0227] From the results in Table 8.2 it appears that lipase 3 improvedthe bread crumb homogeneity and that the average diameter of the crumbpores was reduced significantly. The gluten index also clearlycorrelated to the addition of lipase 3 as an indication of a more firmgluten caused by the modification of the wheat lipid components causingbetter dough stability and a more homogeneous bread pore structure.However, these modifications appeared to be optimal at the addition of5,000 LUS/kg flour of lipase 3 whereas a higher dosage resulted in a toostrong modification of the wheat gluten.

[0228] The results of the GLC and HPLC analyses (Table 8.3) clearlydemonstrated that the triglycerides in the dough were hydrolysed. Butmore interestingly, there was also observed a modification of theglycolipids, monogalactosyl diglyceride and digalactosyl diglyceride.These components were converted to the more polar componentsmonogalactosyl monoglyceride and digalactosyl monoglyceride. Asdigalactosyl monoglyceride is a more surface active component thandigalactosyl diglyceride it is assumed that this component contributedto the observed improved crumb cell structure and homogeneity. It alsoappeared that phospholipids like phosphatidyl choline were only modifiedto a very small extent.

[0229] 8.3. Baking experiments with lipase 3 in Danish rolls

[0230] The effect of adding lipase 3 to a dough for making Danish rollswas evaluated. The enzyme was added as a freeze-dried preparation onmaltodextrin together with the other ingredients. The results of thebaking tests are shown in Tables 8.5 to 8.7. TABLE 8.5 Lipase 3 010,000   20,000   30,000   LUS/kg flour Specific volume of bread 6.86   7.04    6.35    6.36 (45 min fermentation) Specific volume of bread8.30    8.59    8.23    8.04 (65 min fermentation) Subjective evaluationof 3  5  4  4 crumb homogeneity

[0231] TABLE 8.6 Lipase 3 LUS/kg flour 0 10,000 20,000 30,000 Free fattyacids, % 0.060 0.126 0.173 0.211 Monoglycerides, % 0.028 0.050 0.0540.063 Diglycerides, % 0.103 0.095 0.110 0.104 Triglycerides, % 0.7050.561 0.472 0.436

[0232] TABLE 8.7 Lipase 3 LUS/kg flour 0 5,000 15,000 25,000Digalactosyl 0.204 0.187 0.154 0.110 Diglyceride, % Digalactosyl 0.0070.026 0.047 0.074 Monoglyceride, % Phosphatidyl 0.077 0.078 0.077 0.063choline, % Lysophosphatidyl 0.153 0.161 0.162 0.150 choline, %

[0233] It is apparent from the results shown in Table 8.5 that theaddition of lipase 3 does not significantly increase the volume of therolls. Furthermore, lipase 3 was found to improve the homogeneity of thecrumb.

[0234] The GLC and HPLC analyses of the wheat lipids, as shown in Tables8.6 and 8.7, demonstrated the modification of these lipids.

EXAMPLE 9 Dough Improving Effect of Glycerol Oxidase and Lipase

[0235] The effect of glycerol oxidase and lipase (separately or incombination) on dough strength was studied in a dough prepared accordingto the AACC Method 54-10. The dough was subjected to extensiographmeasurements (Barbender Extensiograph EXEK/6) also according to AACCMethod 54-10 with and with out the addition of glycerol oxidase fromAspergillus japonicus combined with lipase from Aspergillus oryzae(GRINDAMYL™ EXEL 16, Bakery Enzyme, Danisco Ingredients). The dough without addition of enzymes served as a control.

[0236] The principle of the above method is that the dough after formingis subjected to a load-extension test after resting at 30° C. for 45, 90and 135 minutes, respectively, using an extensigraph capable ofrecording a load-extension curve (extensigram) which is an indication ofthe doughs resistance to physical deformation when stretched. From thiscurve, the resistance to extension, B (height of curve) and theextensibility, C (total length of curve) can be calculated. The B/Cratio (D) is an indication of the baking strength of the flour dough.The results of the experiment are summarized in Table 9.1 below. TABLE9.1 Extensigraph measurements of dough supplemented with glyceroloxidase and lipase Resting Sample time (per kg flour) (min) B-valueC-value D = B/C Control 45 220 192 1.15  500 LUS lipase 45 225 190 1.181000 U glycerol oxidase 45 300 195 1.54  500 LUS lipase + 45 350 1981.77 1000 U Glycerol oxidase Control 90 240 196 1.22  500 LUS lipase 90245 195 1.16 1000 U Glycerol oxidase 90 330 190 1.74  500 LUS lipase +90 380 192 1.98 1000 U Glycerol oxidase Control 135  260 188 1.38  500LUS lipase 135  265 190 1.39 1000 U Glycerol oxidase 135  380 188 2.02 500 LUS lipase + 135  410 190 2.15 1000 U Glycerol oxidase

[0237] When the results from the above experiments are compared withregard to the differences between the control dough and the glyceroloxidase supplemented dough it appears that glycerol oxidase clearly hasa strengthening effect. The B/C ratio was increased by 34%, 43% and 46%after 45, 90 and 135 minutes of resting time respectively.

[0238] The addition of lipase only did not have any effect on the B/Cratio.

[0239] However, when supplementing the dough with a combination ofglycerol oxidase and lipase, a further increase in the B/C ratio wasseen as compared to bread prepared from dough supplemented with glyceroloxidase only. The B/C ratio was increased by 54%, 62% and 56% after 45,90 and 135 minutes respectively. This clearly indicates that thecombined use of these two enzymes in the preparation of bread productshas an enhancing effect on the baking strength.

EXAMPLE 10 Improvement of the Specific Volume of Bread Prepared fromDough Supplemented with Glycerol Oxidase and Lipase

[0240] The effect of using glycerol oxidase and lipase (separately or incombination) on the specific bread volume and the crumb homogeneity wastested in a baking procedure for Danish rolls with a dough prepared asdescribed in example 8. Glycerol oxidase from Aspergillus japonicus andlipase 3 from Aspergillus tubigensis was added to the dough in differentamounts. Dough without the addition of enzymes served as control. Thefully proofed dough was baked at 220° C. for 18 minutes with 12 secondssteam in a Bago-oven. After cooling the rolls were weighed and thevolume of the rolls were measured by the rape seed displacement method.The specific bread volume was determined as the volume of the bread (ml)divided by the weight of the bread (g). The crumb homogeneity wasevaluated subjectively on a scale from 1 to 7, where 1=courseinhomogeneous and 7=nice homogeneous. The results from this experimentare summarized in Table 10.1 below. TABLE 10.1 Specific volume and crumbhomogeneity in bread supplemented with lipase and glycerol SampleSpecific Crumb homo- (per kg flour) volume (ml/g) geneity Control 5.45 1 1,000 U glycerol oxidase 6.75 2 10,000 LUS lipase 5.65 4 10,000 LUSlipase + 7.25 7  1,000 U glycerol oxidase

[0241] As can be seen in the above Table 10.1, the use of glyceroloxidase in the preparing of bread, significantly increased the breadvolume (24%) as compared to bread prepared from a similar dough notsupplemented with this enzyme. Addition of glycerol oxidase did notimprove the crumb homogeneity significantly.

[0242] The use of lipase in the preparing of bread did not increase thespecific volume of the bread, however a highly increased porehomogeneity was observed.

[0243] The combined use of glycerol oxidase and lipase increased thespecific volume of the bread with 33% as compared to bread prepared froma similar dough not supplemented with any of the two enzymes.

[0244] In addition, the crumb homogeneity was highly improved by thecombined use of lipase and glycerol oxidase as compared to the controlbread and the breads prepared from dough supplemented with lipase andglycerol oxidase respectively.

[0245] This clearly indicates that the combination of lipase andglycerol oxidase in the preparation of bread has a synergistic effectand significantly enhances the shape and appearance of the finishedbread product.

EXAMPLE 11 Hydrolysis of Triglycerides and Formation of Glycerol inDough Supplemented with Lipase

[0246] In order to study the hydrolysis of triglycerides and theformation of glycerol in a proofed dough supplemented with lipase, adough for Danish rolls was prepared in the same manner as described inexample 8. Different amounts of lipase (GRINDAMYL™ EXEL 16) was added tothe dough, and the total lipid from the fully proofed dough wasextracted and analyzed by gas chromatography as described above. TABLE11.1 Triglycerides and glycerol in a dough as a func- tion of lipaseaddition Lipase addition (GRINDAMYL ™ EXEL 16) Glycerol Triglycerides(LUS per kg flour) (%) (%)    0 2.2 7.88   500 2.2 6.22 1,250 2.4 5.992,500 2.8 5.37 3,750 2.9 5.47 5,000 3.0 5.55 7,500 3.1 5.03 10,000  3.04.39

[0247] From the above experiment it is clear that the addition of lipaseto a dough has a hydrolyzing effect on the triglycerides present in thedough, which is seen as a decrease in the triglyceride content asfunction of the increased lipase addition. The resulting level ofglycerol increases as a function of the lipase addition. These resultssuggests, that the improvement of the B/C ratio and the specific breadvolume in bread prepared from dough supplemented with both glyceroloxidase and lipase, as was seen in example 9 and 10, could be due tothat lipase addition to a dough is generating glycerol which further canact as substrate for glycerol oxidase.

SUMMARY PARAGRAPHS

[0248] The present invention is defined in the claims and theaccompanying description.

[0249] For convenience other aspects of the present invention arepresented herein by way of numbered paragraphs.

[0250] 1. A method of improving the rheological properties of a flourdough and the quality of the finished product made from the dough,comprising adding to the dough 10 to 10,000 units of a glycerol oxidaseper kg of flour.

[0251] 2. A method according to paragraph 1 wherein the glycerol oxidaseis derived from an organism selected from the group consisting of abacterial species, a fungal species, a yeast species, an animal cell anda plant cell.

[0252] 3. A method according to paragraph 2 wherein the fungal speciesis selected from the group consisting of an Aspergillus species, aNeurospora species and a Penicillium species.

[0253] 4. A method according to paragraph 1 wherein the resistance toextension of the dough in terms of the ratio between resistance toextension (height of curve, B) and the extensibility (length of curve,C), i.e. the B/C ratio, as measured by the AACC method 54-10 isincreased by at least 10% relative to that of an otherwise similar doughnot containing glycerol oxidase.

[0254] 5. A method according to paragraph 1 wherein the finished productis selected from the group consisting of a bread product, a noodleproduct and an alimentary paste product.

[0255] 6. A method according to paragraph 1 where at least one furtherenzyme is added to the dough ingredients, dough additives or the dough.

[0256] 7. A method according to paragraph 6 wherein the further enzymeis selected from the group consisting of a cellulase, a hemicellulase, astarch degrading enzyme, an oxidoreductase, a lipase and a protease.

[0257] 8. A method of improving the rheological properties of a flourdough and the quality of the finished product made from the dough,comprising adding to the dough a glycerol oxidase and a lipase.

[0258] 9. A method according to paragraph 8 wherein the amount ofglycerol oxidase is in the range of 10 to 10,000 units per kg flour.

[0259] 10. A method according to paragraph 8 wherein the glyceroloxidase is derived from an organism selected from the group consistingof a bacterial species, a fungal species, a yeast species, an animalcell and a plant cell.

[0260] 11. A method according to paragraph 10 wherein the fungal speciesis selected from the group consisting of an Aspergillus species, aNeurospora species and a Penicillium species.

[0261] 12. A method according to paragraph 8 wherein the resistance toextension of the dough in terms of the ratio between resistance toextension (height of curve, B) and the extensibility (length of curve,C), i.e. the B/C ratio, as measured by the AACC method 54-10 isincreased by at least 10% relative to that of an otherwise similar doughnot containing glycerol oxidase.

[0262] 13. A method according to paragraph 8 wherein the finishedproduct is selected from the group consisting of a bread product, anoodle product and an alimentary paste product.

[0263] 14. A method according to paragraph 8 where at least one furtherenzyme is added to the dough ingredients, dough additives or the dough.

[0264] 15. A method according to paragraph 14 wherein the further enzymeis selected from the group consisting of a cellulase, a hemicellulase, astarch degrading enzyme, an oxidoreductase, and a protease.

[0265] 16. A method according to paragraph 8 wherein the amount oflipase is in the range of 10 to 100,000 LUS per kg of flour.

[0266] 17. A method according to paragraph 8 wherein the lipase isderived from an organism selected from the group consisting of abacterial species, a fungal species, a yeast species, an animal cell anda plant cell.

[0267] 18. A method according to paragraph 17 wherein the lipase isderived from an Aspergillus species.

[0268] 19. A method according to paragraph 18 wherein the Aspergillusspecies is selected from the group consisting of A. tubigensis, A.oryzae and A. niger.

[0269] 20. A method according to paragraph 8 wherein at least 10% of thegalactosyl diglycerides normally present in a flour dough is hydrolysedto the corresponding galactosyl monoglycerides.

[0270] 21. A dough improving composition comprising a glycerol oxidaseand at least one further dough ingredient or dough additive.

[0271] 22. A composition according to paragraph 21 wherein the furtherdough additive is selected from the group consisting of a substrate forglycerol oxidase and a lipase.

[0272] 23. A composition according to paragraph 22 which is apre-mixture useful for preparing a baked product or in making a noodleproduct or an alimentary paste product.

[0273] 24. A composition according to paragraph 21 which comprises anadditive selected from the group consisting of an emulsifying agent anda hydrocolloid.

[0274] 25. A composition according to paragraph 24 wherein thehydrocolloid is selected from the group consisting of an alginate, acarrageenan, a pectin and a vegetable gum.

[0275] 26. A composition according to paragraph 21 wherein the amount ofglycerol oxidase is in the range of 10 to 10,000 units per kg flour.

[0276] 27. A composition according to paragraph 21 or 26, comprising asthe further dough additive a lipase in an amount which is in the rangeof 10 to 100,000 LUS per kg flour.

[0277] 28. Use of a glycerol oxidase for improving the rheologicalproperties of a flour dough and the quality of the finished product madefrom the dough.

[0278] 29. Use according to paragraph 28 wherein the improvement of therheological properties include that the resistance to extension of thedough in terms of the ratio between resistance to extension (height ofcurve, B) and the extensibility (length of curve, C), i.e. the B/Cratio, as measured by the AACC method 54-10 is increased by at least 10%relative to that of an otherwise similar dough not containing glyceroloxidase.

[0279] 30. Use of a glycerol oxidase and a lipase in combination forimproving the rheological properties of a flour dough and the quality ofthe finished product made from the dough.

[0280] 31. Use according to paragraph 30 wherein the improvement of therheological properties of the dough include that the resistance toextension of the dough in terms of the ratio between resistance toextension (height of curve, B) and the extensibility (length of curve,C), i.e. the B/C ratio, as measured by the AACC method 54-10 isincreased by at least 10% relative to that of an otherwise similar doughthat does not contain glycerol oxidase.

[0281] 32. Use according to paragraph 30 wherein the improvement of thequality of the finished product made from the dough is that the averagepore diameter of the crumb of the bread made from the dough is reducedby at least 10%, relative to a bread which is made from a bread doughwithout addition of the lipase.

[0282] 33. Use according to paragraph 30 wherein the improvement of thequality of the finished product made from the dough is that the porehomogeneity of the crumb of the bread made from the dough is increasedby at least 5%, relative to a bread which is made from a bread doughwithout addition of the lipase.

[0283] 34. Use according to paragraph 30 or 31 wherein the improvementof the rheological characteristics of the dough includes that the glutenindex in the dough is increased by at least 5%, relative to a doughwithout addition of a lipase, the gluten index is determined by means ofa Glutomatic 2200 apparatus.

1 22 1 25 PRT Aspergillus tubingensis MISC_FEATURE (22)..(22) “Xaa” canbe any amino acid 1 Ser Val Ser Thr Ser Thr Leu Asp Glu Leu Gln Leu PheAla Gln Trp 1 5 10 15 Ser Ala Ala Ala Tyr Xaa Ser Asn Asn 20 25 2 7 PRTAspergillus tubingensis 2 Val His Thr Gly Phe Trp Lys 1 5 3 14 PRTAspergillus tubingensis 3 Ala Trp Glu Ser Ala Ala Asp Glu Leu Thr SerLys Ile Lys 1 5 10 4 20 DNA Artificial Sequence PCR primer used for PCRamplification of a fragment of the lipase gene 4 ttccaraanc cngtrtgnac20 5 18 DNA Artificial Sequence PCR primer used for PCR amplification ofa fragment of the lipase gene 5 carytnttyg cncartgg 18 6 17 DNAArtificial Sequence PCR primer used for PCR amplification of a fragmentof the lipase gene 6 gcvgchswyt cccavgc 17 7 317 DNA Aspergillustubingensis 7 cagttgttcg cgcaatggtc tgccgcagct tattgctcga ataatatcgactcgaaagav 60 tccaacttga catgcacggc caacgcctgt ccatcagtcg aggaggccagtaccacgatg 120 ctgctggagt tcgacctgta tgtcactcag atcgcagaca tagagcacagctaattgaac 180 aggacgaacg acttttggag gcacagccgg tttcctggcc gcggacaacaccaacaagcg 240 gctcgtggtc gccttccggg gaagcagcac gattgagaac tggattgctaatcytgactt 300 catcctggra gataacg 317 8 1045 DNA Aspergillus tubingensis8 atgttctctg gacggtttgg agtgcttttg acagcgcttg ctgcgctggg tgctgccgcg 60ccggcaccgc ttgctgtgcg gagtaggtgt gcccgatgtg agatggttgg atagcactga 120tgaagggtga ataggtgtct cgacttccac gttggatgag ttgcaattgt tcgcgcaatg 180gtctgccgca gcttattgct cgaataatat cgactcgaaa gactccaact tgacatgcac 240ggccaacgcc tgtccatcag tcgaggaggc cagtaccacg atgctgctgg agttcgacct 300gtatgtcact cagatcgcag acatagagca cagctaattt gaacaggacg aacgactttg 360gaggcacagc cggtttcctg gccgcggaca acaccaacaa gcggctcgtg gtcgccttcc 420ggggaagcag cacgattgag aactggattg ctaatcttga cttcatcctg gaagataacg 480acgacctctg caccggctgc aaggtccata ctggtttctg gaaggcatgg gagtccgctg 540ccgacgaact gacgagcaag atcaagtctg cgatgagcac gtattcgggc tataccctat 600acttcaccgg gcacagtttg ggcggcgcat tggctacgct gggagcgaca gttctgcgaa 660atgacggata tagcgttgag ctggtgagtc cttcacaaag gtgatggagc gacaatcggg 720aacagacagt caatagtaca cctatggatg tcctcgaatc ggaaactatg cgctggctga 780gcatatcacc agtcagggat ctggggccaa cttccgtgtt acacacttga acgacatcgt 840cccccgggtg ccacccatgg actttggatt cagtcagcca agtccggaat actggatcac 900cagtggcaat ggagccagtg tcacggcgtc ggatatcgaa gtcatcgagg gaatcaattc 960aacggcggga aatgcaggcg aagcaacggt gagcgttgtg gctcacttgt ggtacttttt 1020tgcgatttcc gagtgcctgc tataa 1045 9 297 PRT Aspergillus tubingensis 9 MetPhe Ser Gly Arg Phe Gly Val Leu Leu Thr Ala Leu Ala Ala Leu 1 5 10 15Gly Ala Ala Ala Pro Ala Pro Leu Ala Val Arg Ser Val Ser Thr Ser 20 25 30Thr Leu Asp Glu Leu Gln Leu Phe Ala Gln Trp Ser Ala Ala Ala Tyr 35 40 45Cys Ser Asn Asn Ile Asp Ser Lys Asp Ser Asn Leu Thr Cys Thr Ala 50 55 60Asn Ala Cys Pro Ser Val Glu Glu Ala Ser Thr Thr Met Leu Leu Glu 65 70 7580 Phe Asp Leu Thr Asn Asp Phe Gly Gly Thr Ala Gly Phe Leu Ala Ala 85 9095 Asp Asn Thr Asn Lys Arg Leu Val Val Ala Phe Arg Gly Ser Ser Thr 100105 110 Ile Glu Asn Trp Ile Ala Asn Leu Asp Phe Ile Leu Glu Asp Asn Asp115 120 125 Asp Leu Cys Thr Gly Cys Lys Val His Thr Gly Phe Trp Lys AlaTrp 130 135 140 Glu Ser Ala Ala Asp Glu Leu Thr Ser Lys Ile Lys Ser AlaMet Ser 145 150 155 160 Thr Tyr Ser Gly Tyr Thr Leu Tyr Phe Thr Gly HisSer Leu Gly Gly 165 170 175 Ala Leu Ala Thr Leu Gly Ala Thr Val Leu ArgAsn Asp Gly Tyr Ser 180 185 190 Val Glu Leu Tyr Thr Tyr Gly Cys Pro ArgIle Gly Asn Tyr Ala Leu 195 200 205 Ala Glu His Ile Thr Ser Gln Gly SerGly Ala Asn Phe Arg Val Thr 210 215 220 His Leu Asn Asp Ile Val Pro ArgVal Pro Pro Met Asp Phe Gly Phe 225 230 235 240 Ser Gln Pro Ser Pro GluTyr Trp Ile Thr Ser Gly Asn Gly Ala Ser 245 250 255 Val Thr Ala Ser AspIle Glu Val Ile Glu Gly Ile Asn Ser Thr Ala 260 265 270 Gly Asn Ala GlyGlu Ala Thr Val Ser Val Val Ala His Leu Trp Tyr 275 280 285 Phe Phe AlaIle Ser Glu Cys Leu Leu 290 295 10 392 PRT Rhizopus delamar 10 Met ValSer Phe Ile Ser Ile Ser Gln Gly Val Ser Leu Cys Leu Leu 1 5 10 15 ValSer Ser Met Met Leu Gly Ser Ser Ala Val Pro Val Ser Gly Lys 20 25 30 SerGly Ser Ser Asn Thr Ala Val Ser Ala Ser Asp Asn Ala Ala Leu 35 40 45 ProPro Leu Ile Ser Ser Arg Cys Ala Pro Pro Ser Asn Lys Gly Ser 50 55 60 LysSer Asp Leu Gln Ala Glu Pro Tyr Asn Met Gln Lys Asn Thr Glu 65 70 75 80Trp Tyr Glu Ser His Gly Gly Asn Leu Thr Ser Ile Gly Lys Arg Asp 85 90 95Asp Asn Leu Val Gly Gly Met Thr Leu Asp Leu Pro Ser Asp Ala Pro 100 105110 Pro Ile Ser Leu Ser Ser Ser Thr Asn Ser Ala Ser Asp Gly Gly Lys 115120 125 Val Val Ala Ala Thr Thr Ala Gln Ile Gln Glu Phe Thr Lys Tyr Ala130 135 140 Gly Ile Ala Ala Thr Ala Tyr Cys Arg Ser Val Val Pro Gly AsnLys 145 150 155 160 Trp Asp Cys Val Gln Cys Gln Lys Trp Val Pro Asp GlyLys Ile Ile 165 170 175 Thr Thr Phe Thr Ser Leu Leu Ser Asp Thr Asn GlyTyr Val Leu Arg 180 185 190 Ser Asp Lys Gln Lys Thr Ile Tyr Leu Val PheArg Gly Thr Asn Ser 195 200 205 Phe Arg Ser Ala Ile Thr Asp Ile Val PheAsn Phe Ser Asp Tyr Lys 210 215 220 Pro Val Lys Gly Ala Lys Val His AlaGly Phe Leu Ser Ser Tyr Glu 225 230 235 240 Gln Val Val Asn Asp Tyr PhePro Val Val Gln Glu Gln Leu Thr Ala 245 250 255 His Pro Thr Tyr Lys ValIle Val Thr Gly His Ser Leu Gly Gly Ala 260 265 270 Gln Ala Leu Leu AlaGly Met Asp Leu Tyr Gln Arg Glu Pro Arg Leu 275 280 285 Ser Pro Lys AsnLeu Ser Ile Phe Thr Val Gly Gly Pro Arg Val Gly 290 295 300 Asn Pro ThrPhe Ala Tyr Tyr Val Glu Ser Thr Gly Ile Pro Phe Gln 305 310 315 320 ArgThr Val His Lys Arg Asp Ile Val Pro His Val Pro Pro Gln Ser 325 330 335Phe Gly Phe Leu His Pro Gly Val Glu Ser Trp Ile Lys Ser Gly Thr 340 345350 Ser Asn Val Gln Ile Cys Thr Ser Glu Ile Glu Thr Lys Asp Cys Ser 355360 365 Asn Ser Ile Val Pro Phe Thr Ser Ile Leu Asp His Leu Ser Tyr Phe370 375 380 Asp Ile Asn Glu Gly Ser Cys Leu 385 390 11 363 PRTRhizomucor miehei 11 Met Val Leu Lys Gln Arg Ala Asn Tyr Leu Gly Phe LeuIle Val Phe 1 5 10 15 Phe Thr Ala Phe Leu Val Glu Ala Val Pro Ile LysArg Gln Ser Asn 20 25 30 Ser Thr Val Asp Ser Leu Pro Pro Leu Ile Pro SerArg Thr Ser Ala 35 40 45 Pro Ser Ser Ser Pro Ser Thr Thr Asp Pro Glu AlaPro Ala Met Ser 50 55 60 Arg Asn Gly Pro Leu Pro Ser Asp Val Glu Thr LysTyr Gly Met Ala 65 70 75 80 Leu Asn Ala Thr Ser Tyr Pro Asp Ser Val ValGln Ala Met Ser Ile 85 90 95 Asp Gly Gly Ile Arg Ala Ala Thr Ser Gln GluIle Asn Glu Leu Thr 100 105 110 Tyr Tyr Thr Thr Leu Ser Ala Asn Ser TyrCys Arg Thr Val Ile Pro 115 120 125 Gly Ala Thr Trp Asp Cys Ile His CysAsp Ala Thr Glu Asp Leu Lys 130 135 140 Ile Ile Lys Thr Trp Ser Thr LeuIle Tyr Asp Thr Asn Ala Met Val 145 150 155 160 Ala Arg Gly Asp Ser GluLys Thr Ile Tyr Ile Val Phe Arg Gly Ser 165 170 175 Ser Ser Ile Arg AsnTrp Ile Ala Asp Leu Thr Phe Val Pro Val Ser 180 185 190 Tyr Pro Pro ValSer Gly Thr Lys Val His Lys Gly Phe Leu Asp Ser 195 200 205 Tyr Gly GluVal Gln Asn Glu Leu Val Ala Thr Val Leu Asp Gln Phe 210 215 220 Lys GlnTyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser Leu Gly 225 230 235 240Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg Glu Glu 245 250255 Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln Pro Arg 260265 270 Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro275 280 285 Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu ProPro 290 295 300 Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp IleThr Asp 305 310 315 320 Asn Ser Pro Glu Thr Val Gln Val Cys Thr Ser AspLeu Glu Thr Ser 325 330 335 Asp Cys Ser Asn Ser Ile Val Pro Phe Thr SerVal Leu Asp His Leu 340 345 350 Ser Tyr Phe Gly Ile Asn Thr Gly Leu CysThr 355 360 12 305 PRT Penicillium camemberti 12 Met Arg Leu Ser Phe PheThr Ala Leu Ser Ala Val Ala Ser Leu Gly 1 5 10 15 Tyr Ala Leu Pro GlyLys Leu Gln Ser Arg Asp Val Ser Thr Ser Glu 20 25 30 Leu Asp Gln Phe GluPhe Trp Val Gln Tyr Ala Ala Ala Ser Tyr Tyr 35 40 45 Glu Ala Asp Tyr ThrAla Gln Val Gly Asp Lys Leu Ser Cys Ser Lys 50 55 60 Gly Asn Cys Pro GluVal Glu Ala Thr Gly Ala Thr Val Ser Tyr Asp 65 70 75 80 Phe Ser Asp SerThr Ile Thr Asp Thr Ala Gly Tyr Ile Ala Val Asp 85 90 95 His Thr Asn SerAla Val Val Leu Ala Phe Arg Gly Ser Tyr Ser Val 100 105 110 Arg Asn TrpVal Ala Asp Ala Thr Phe Val His Thr Asn Pro Gly Leu 115 120 125 Cys AspGly Cys Leu Ala Glu Leu Gly Phe Trp Ser Ser Trp Lys Leu 130 135 140 ValArg Asp Asp Ile Ile Lys Glu Leu Lys Glu Val Val Ala Gln Asn 145 150 155160 Pro Asn Tyr Glu Leu Val Val Val Gly His Ser Leu Gly Ala Ala Val 165170 175 Ala Thr Leu Ala Ala Thr Asp Leu Arg Gly Lys Gly Tyr Pro Ser Ala180 185 190 Lys Leu Tyr Ala Tyr Ala Ser Pro Arg Val Gly Asn Ala Ala LeuAla 195 200 205 Lys Tyr Ile Thr Ala Gln Gly Asn Asn Phe Arg Phe Thr HisThr Asn 210 215 220 Asp Pro Val Pro Lys Leu Pro Leu Leu Ser Met Gly TyrVal His Val 225 230 235 240 Ser Pro Glu Tyr Trp Ile Thr Ser Pro Asn AsnAla Thr Val Ser Thr 245 250 255 Ser Asp Ile Lys Val Ile Asp Gly Asp ValSer Phe Asp Gly Asn Thr 260 265 270 Gly Thr Gly Leu Pro Leu Leu Thr AspPhe Glu Ala His Ile Trp Tyr 275 280 285 Phe Val Gln Val Asp Ala Gly LysGly Pro Gly Leu Pro Phe Lys Arg 290 295 300 Val 305 13 334 DNAAspergillus tubingensis misc_feature (10)..(10) “n” can be a or g or cor t/u 13 tacccggggn tccgatt cag ttg ttc gcg caa tgg tct gcc gca gct tat50 Gln Leu Phe Ala Gln Trp Ser Ala Ala Ala Tyr 1 5 10 tgc tcg aat aatatc gac tcg aaa gav tcc aac ttg aca tgc acg gcc 98 Cys Ser Asn Asn IleAsp Ser Lys Xaa Ser Asn Leu Thr Cys Thr Ala 15 20 25 aac gcc tgt cca tcagtc gag gag gcc agt acc acg atg ctg ctg gag 146 Asn Ala Cys Pro Ser ValGlu Glu Ala Ser Thr Thr Met Leu Leu Glu 30 35 40 ttc gac ctg tat gtc actcag atc gca gac ata gag cac agc taa ttg 194 Phe Asp Leu Tyr Val Thr GlnIle Ala Asp Ile Glu His Ser Leu 45 50 55 aac agg acg aac gac ttt tgg aggcac agc cgg ttt cct ggc cgc gga 242 Asn Arg Thr Asn Asp Phe Trp Arg HisSer Arg Phe Pro Gly Arg Gly 60 65 70 caa cac caa caa gcg gct cgt ggt cgcctt ccg ggg aag cag cac gat 290 Gln His Gln Gln Ala Ala Arg Gly Arg LeuPro Gly Lys Gln His Asp 75 80 85 90 tga gaa ctg gat tgc taa tcy tga cttcat cct ggr aga taacg 334 Glu Leu Asp Cys Xaa Leu His Pro Xaa Arg 95 10014 57 PRT Aspergillus tubingensis misc_feature (20)..(20) The ′Xaa′ atlocation 20 stands for Glu, or Asp. 14 Gln Leu Phe Ala Gln Trp Ser AlaAla Ala Tyr Cys Ser Asn Asn Ile 1 5 10 15 Asp Ser Lys Xaa Ser Asn LeuThr Cys Thr Ala Asn Ala Cys Pro Ser 20 25 30 Val Glu Glu Ala Ser Thr ThrMet Leu Leu Glu Phe Asp Leu Tyr Val 35 40 45 Thr Gln Ile Ala Asp Ile GluHis Ser 50 55 15 33 PRT Aspergillus tubingensis misc_feature (10)..(10)“n” can be a or g or c or t/u 15 Leu Asn Arg Thr Asn Asp Phe Trp Arg HisSer Arg Phe Pro Gly Arg 1 5 10 15 Gly Gln His Gln Gln Ala Ala Arg GlyArg Leu Pro Gly Lys Gln His 20 25 30 Asp 16 4 PRT Aspergillustubingensis misc_feature (10)..(10) “n” can be a or g or c or t/u 16 GluLeu Asp Cys 1 17 5 PRT Aspergillus tubingensis misc_feature (4)..(4) The′Xaa′ at location 4 stands for Gly. 17 Leu His Pro Xaa Arg 1 5 18 1833DNA Aspergillus tubingensis misc_feature (3)..(3) n can be a or g or cor t/u 18 ccndttaatc ccccaccggg gttcccgctc ccggatggag atggggccaaaactggcaac 60 ccccagttgc gcaacggaac aaccgccgac ccggaacaaa ggatgcggatgaggagatac 120 ggtgcctgat tgcatggctg gcttcatctg ctatcgtgac agtgctctttgggtgaatat 180 tgttgtctga cttaccccgc ttcttgcttt ttcccccctg aggccctgatggggaatcgc 240 ggtgggtaat atgatatggg tataaaaggg agatcggagg tgcagttggattgaggcagt 300 gtgtgtgtgt gcattgcaga agcccgttgg tcgcaaggtt ttggtcgcctcgattgtttg 360 tataccgcaa g atg ttc tct gga cgg ttt gga gtg ctt ttg acagcg ctt 410 Met Phe Ser Gly Arg Phe Gly Val Leu Leu Thr Ala Leu 1 5 10gct gcg ctg ggt gct gcc gcg ccg gca ccg ctt gct gtg cgg a 453 Ala AlaLeu Gly Ala Ala Ala Pro Ala Pro Leu Ala Val Arg 15 20 25 gtaggtgtgcccgatgtgag atggttggat agcactgatg aagggtgaat ag gt gtc 510 Ser Val tcgact tcc acg ttg gat gag ttg caa ttg ttc gcg caa tgg tct gcc 558 Ser ThrSer Thr Leu Asp Glu Leu Gln Leu Phe Ala Gln Trp Ser Ala 30 35 40 45 gcagct tat tgc tcg aat aat atc gac tcg aaa gac tcc aac ttg aca 606 Ala AlaTyr Cys Ser Asn Asn Ile Asp Ser Lys Asp Ser Asn Leu Thr 50 55 60 tgc acggcc aac gcc tgt cca tca gtc gag gag gcc agt acc acg atg 654 Cys Thr AlaAsn Ala Cys Pro Ser Val Glu Glu Ala Ser Thr Thr Met 65 70 75 ctg ctg gagttc gac ctg tatgtcactc agatcgcaga catagagcac 702 Leu Leu Glu Phe Asp Leu80 agctaatttg aacagg acg aac gac ttt gga ggc aca gcc ggt ttc ctg gcc 754Thr Asn Asp Phe Gly Gly Thr Ala Gly Phe Leu Ala 85 90 95 gcg gac aac accaac aag cgg ctc gtg gtc gcc ttc cgg gga agc agc 802 Ala Asp Asn Thr AsnLys Arg Leu Val Val Ala Phe Arg Gly Ser Ser 100 105 110 acg att gag aactgg att gct aat ctt gac ttc atc ctg gaa gat aac 850 Thr Ile Glu Asn TrpIle Ala Asn Leu Asp Phe Ile Leu Glu Asp Asn 115 120 125 gac gac ctc tgcacc ggc tgc aag gtc cat act ggt ttc tgg aag gca 898 Asp Asp Leu Cys ThrGly Cys Lys Val His Thr Gly Phe Trp Lys Ala 130 135 140 tgg gag tcc gctgcc gac gaa ctg acg agc aag atc aag tct gcg atg 946 Trp Glu Ser Ala AlaAsp Glu Leu Thr Ser Lys Ile Lys Ser Ala Met 145 150 155 agc acg tat tcgggc tat acc cta tac ttc acc ggg cac agt ttg ggc 994 Ser Thr Tyr Ser GlyTyr Thr Leu Tyr Phe Thr Gly His Ser Leu Gly 160 165 170 175 ggc gca ttggct acg ctg gga gcg aca gtt ctg cga aat gac gga tat 1042 Gly Ala Leu AlaThr Leu Gly Ala Thr Val Leu Arg Asn Asp Gly Tyr 180 185 190 agc gtt gagctg gtgagtcctt cacaaaggtg atggagcgac aatcgggaac 1094 Ser Val Glu Leu 195agacagtcaa tag tac acc tat gga tgt cct cga atc gga aac tat gcg 1143 TyrThr Tyr Gly Cys Pro Arg Ile Gly Asn Tyr Ala 200 205 ctg gct gag cat atcacc agt cag gga tct ggg gcc aac ttc cgt gtt 1191 Leu Ala Glu His Ile ThrSer Gln Gly Ser Gly Ala Asn Phe Arg Val 210 215 220 aca cac ttg aac gacatc gtc ccc cgg gtg cca ccc atg gac ttt gga 1239 Thr His Leu Asn Asp IleVal Pro Arg Val Pro Pro Met Asp Phe Gly 225 230 235 ttc agt cag cca agtccg gaa tac tgg atc acc agt ggc aat gga gcc 1287 Phe Ser Gln Pro Ser ProGlu Tyr Trp Ile Thr Ser Gly Asn Gly Ala 240 245 250 255 agt gtc acg gcgtcg gat atc gaa gtc atc gag gga atc aat tca acg 1335 Ser Val Thr Ala SerAsp Ile Glu Val Ile Glu Gly Ile Asn Ser Thr 260 265 270 gcg gga aat gcaggc gaa gca acg gtg agc gtt gtg gct cac ttg tgg 1383 Ala Gly Asn Ala GlyGlu Ala Thr Val Ser Val Val Ala His Leu Trp 275 280 285 tac ttt ttt gcgatt tcc gag tgc ctg cta taactagacc gactgtcaga 1433 Tyr Phe Phe Ala IleSer Glu Cys Leu Leu 290 295 ttagtggacg ggagaagtgt acataagtaa ttagtatataatcagagcaa cccagtggtg 1493 gtgatggtgg tgaaagaaga aacacattga gttcccattacgkagcagwt aaagcacktk 1553 kggaggcgct ggttcctcca cttggcagtt ggcggccatcaatcatcttt cctctcctta 1613 ctttcgtcca ccacaactcc catcctgcca gctgtcgcatccccgggttg caacaactat 1673 cgcctccggg gcctccgtgg ttctcctata ttattccatccgacggccga cgtttcaccc 1733 tcaacctgcg ccgccgcaaa atctccccga gtcggtcaactccctcgaac cgccgcccgc 1793 atcgacctca cgaccccgac cgtctgygat ygtccaaccg1833 19 14 PRT Artificial Sequence selected lipase 3 peptide 19 Ala TrpGlu Ser Ala Ala Asp Glu Leu Thr Ser Lys Ile Lys 1 5 10 20 25 PRTArtificial Sequence N terminal lipase 3 peptide 20 Ser Val Ser Thr SerThr Leu Asp Glu Leu Gln Leu Phe Ala Gln Trp 1 5 10 15 Ser Ala Ala AlaTyr Xaa Ser Asn Asn 20 25 21 6 PRT Artificial Sequence portion ofN-terminal lipase peptide used in synthesizing PCR primer C036 21 GlnLeu Phe Ala Gln Trp 1 5 22 6 PRT Artificial Sequence portion ofN-terminal lipase peptide used in synthesizing PCR primer C037 22 AlaTrp Glu Ser Ala Ala 1 5

We claim:
 1. A polypeptide having lipase activity; wherein saidpolypeptide is a triacylglycerol hydrolysing enzyme; and wherein saidpolypeptide is capable of hydrolysing glycolipids that are normallypresent in a flour to the corresponding galactosyl monoglycerides,wherein said polypeptide is capable of hydrolysing at least 10% ofgalactosyl diglycerides normally present in a flour dough tomonoglycerides.
 2. A polypeptide having lipase activity; wherein saidpolypeptide is a triacylglycerol hydrolysing enzyme and wherein saidpolypeptide retains at least 82.5% activity after 4 days at roomtemperature and at a pH in the range of 3.5-8, and wherein saidpolypeptide is capable of hydrolysing glycolipids that are normallypresent in a flour to the corresponding galactosyl monoglycerides,wherein said polypeptide is capable of hydrolysing at least 10% ofgalactosyl diglycerides normally present in a flour dough tomonoglycerides.
 3. A polypeptide having lipase activity; wherein saidpolypeptide is a triacylglycerol hydrolysing enzyme, wherein saidpolypeptide retains at least 82.5% activity after 4 days at roomtemperature and at a pH in the range of 3.5-8.
 4. A polypeptide havinglipase activity; wherein said polypeptide is a triacylglycerolhydrolysing enzyme; and wherein said polypeptide is capable ofhydrolysing glycolipids, monogalactosyl diglyceride and digalactosyldiglyceride, that are normally present in a flour to monogalactosylmonoglyceride and digalactosyl monoglyceride.
 5. A polypeptide havinglipase activity; wherein said polypeptide is a triacylglycerolhydrolysing enzyme and wherein said polypeptide retains at least 82.5%activity after 4 days at room temperature and at a pH in the range of3.5-8, and wherein said polypeptide is capable of hydrolysingglycolipids, monogalactosyl diglyceride and digalactosyl diglyceride,that are normally present in a flour, to the corresponding galactosylmonoglycerides.
 6. A polypeptide having lipase activity; wherein saidpolypeptide is a triacylglycerol hydrolysing enzyme and wherein saidpolypeptide is capable of modifying by hydrolysis the glycolipids,monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) tothe more polar components monogalactosyl monoglyceride (MGMG) anddigalactosyl monoglyceride (DGMG).
 7. A polypeptide having lipaseactivity; wherein said polypeptide is a triacylglycerol hydrolysingenzyme and wherein said polypeptide retains at least 82.5% activityafter 4 days at 20° C. and at a pH in the range of 3.5-8, wherein saidpolypeptide is capable of modifying by hydrolysis the glycolipids,monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG),to the more polar components monogalactosyl monoglyceride (MGMG) anddigalactosyl monoglyceride (DGMG).
 8. A polypeptide having lipaseactivity; wherein said polypeptide is a triacylglycerol hydrolysingenzyme and wherein said polypeptide is capable of hydrolysingglycolipids that are normally present in a flour to galactosylmonoglycerides, wherein said polypeptide is capable of modifying byhydrolysis the glycolipids, monogalactosyl diglyceride (MGDG) anddigalactosyl diglyceride (DGDG) to the more polar componentsmonogalactosyl monoglyceride (MGMG) and digalactosyl monoglyceride(DGMG).
 9. A polypeptide having lipase activity; wherein saidpolypeptide is a triacylglycerol hydrolysing enzyme and wherein saidpolypeptide is capable of hydrolysing galactosyl diglycerides that arenormally present in a flour to galactosyl monoglycerides, wherein saidpolypeptide is capable of hydrolysing at least 10% of the galactosyldiglycerides normally present in a flour dough to monoglycerides,wherein said polypeptide is capable of modifying by hydrolysis thegalactosyl diglycerides, monogalactosyl diglyceride (MGDG) anddigalactosyl diglyceride (DGDG), to the more polar componentsmonogalactosyl monoglyceride (MGMG) and digalactosyl monoglyceride(DGMG).
 10. A polypeptide having lipase activity; wherein saidpolypeptide is a triacylglycerol hydrolysing enzyme and wherein saidpolypeptide retains at least 82.5% activity after 4 days at 20° C. andat a pH in the range of 3.5-8, and wherein said polypeptide is capableof hydrolysing glycolipids that are normally present in a flour togalactosyl monoglycerides, wherein said polypeptide is capable ofmodifying by hydrolysis the glycolipids, monogalactosyl diglyceride(MGDG) and digalactosyl diglyceride (DGDG) to the more polar componentsmonogalactosyl monoglyceride (MGMG) and digalactosyl monoglyceride(DGMG).
 11. A polypeptide having lipase activity; wherein saidpolypeptide is a triacylglycerol hydrolysing enzyme and wherein saidpolypeptide retains at least 82.5% activity after 4 days at 20° C. andat a pH in the range of 3.5-8, and wherein said polypeptide is capableof hydrolysing galactosyl diglycerides that are normally present in aflour to the corresponding galactosyl monoglycerides, wherein saidpolypeptide is capable of hydrolysing at least 10% of the galactosyldiglycerides normally present in a flour dough to the monoglycerides,wherein said polypeptide is capable of modifying by hydrolysis thegalactosyl diglycerides, monogalactosyl diglyceride (MGDG) anddigalactosyl diglyceride (DGDG) to the more polar componentsmonogalactosyl monoglyceride (MGMG) and digalactosyl monoglyceride(DGMG).
 12. A polypeptide having lipase activity; wherein saidpolypeptide is capable of modifying by hydrolysis the glycolipids,monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) tothe more polar components monogalactosyl monoglyceride (MGMG) anddigalactosyl monoglyceride (DGMG).
 13. A polypeptide comprising at leastone amino acid sequence shown herein as SEQ ID NO:1, SEQ ID NO:2 and SEQID NO:3.
 14. A polypeptide according to claim 1 wherein the polypeptideis derivable from Aspergillus tubigensis.
 15. A polypeptide according toclaim 1, wherein the polypeptide has the following characteristics: (i)it retains at least 80% activity after 4 days at 20° C. at a pH in therange of 3.5-8, (ii) it retains at least 60% of its activity after 1hour at 60° C. in 100 mM sodium acetate buffer at pH 5.0, and (iii) ithas an isoelectric point as determined by isoelectric focusing of4.1±0.1.
 16. A polypeptide according to claim 1 wherein said polypeptidecomprises at least one amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO;3, wherein Xaa insaid sequences is an amino acid selected from the group consisting ofAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr and Val.
 17. A polypeptide according to claim 1wherein said lipase has an enzymatic activity at a pH in the range of3.5-8.
 18. A polypeptide according to claim 1, which polypeptide retainsat least 80% of its activity after 1 hour at 50° C. in 100 mM sodiumacetate buffer at pH 5.0.
 19. A polypeptide according to claim 1 whichpolypeptide has an isoelectric point as determined by isoelectricfocusing of 4.1±0.1.
 20. A polypeptide according to claim 1 that iscapable of hydrolysing at least 10% of the galactosyl diglyceridesnormally present in a flour dough to the corresponding galactosylmonoglycerides.
 21. A polypeptide according to claim 1 wherein thepolypeptide is in a substantially purified form.
 22. A polypeptideaccording to claim 1 wherein the polypeptide has a molecular weight asdetermined by matrix-assisted laser desorption ionisation massspectrometry (MALDI-MS) of 31±1.5 kDa.
 23. A polypeptide according toclaim 1 wherein the polypeptide comprises the amino acid sequence shownas SEQ ID NO:9 or a variant, homologue or fragment thereof.
 24. Apolypeptide according to claim 1 wherein the polypeptide is derived froman organism including a fungus, a yeast, a bacterium, a plant cell or ananimal cell.
 25. A polypeptide according to claim 1 wherein when thepolypeptide is added to a bread dough in an amount of 5,000 LUS per kgflour it reduces the average pore diameter of the crumb of the breadmade from the dough by at least 10%, relative to a bread which is madefrom a bread dough without addition of the polypeptide.
 26. Apolypeptide according to claim 1 wherein when it is added to a breaddough in an amount of 5,000 LUS per kg flour, it increases the porehomogeneity of the crumb of the bread made from the dough by at least5%, relative to a bread which is made from a bread dough withoutaddition of the polypeptide.
 27. A polypeptide according to claim 1wherein when it is added to a bread dough in an amount of 5,000 LUS perkg flour, it increases the gluten index in the dough by at least 5%,relative to a dough without addition of the polypeptide, the glutenindex being determined by means of a Glutomatic 2200 apparatus.
 28. Arecombinant DNA molecule comprising a nucleotide sequence coding for thepolypeptide according to claim
 1. 29. A recombinant DNA moleculeaccording to claim 28 comprising at least one of SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 and SEQ ID NO:7.
 30. A recombinant DNA moleculeaccording to claim 28 comprising SEQ ID NO:8 or a variant, homologue orfragment thereof or a sequence complementary thereto.
 31. A recombinantDNA molecule according to claim 28 which is a plasmid deposited underthe accession No. NCIMB
 40863. 32. A cell comprising a recombinant DNAmolecule according to claim 28 and capable of expressing the polypeptideaccording to claim
 1. 33. A cell according to claim 32 which is amicroorganism comprising a fungus, a yeast, a bacterium, a plant cell oran animal cell.
 34. A cell according to claim 33 which is a filamentousfungus comprising an Aspergillus sp., a Penicillium sp., a Rhizomucorsp., or a Neurospora sp.
 35. A cell according to claim 34 which isAspergillus tubigensis.
 36. A method of preparing a polypeptideaccording to claim 1 comprising transforming a host cell with arecombinant DNA molecule according to claim 28, the host cell beingcapable of expressing the nucleotide sequence coding for thepolypeptide, cultivating the transformed host cell under conditionswhere the nucleotide sequence is expressed and harvesting thepolypeptide.
 37. A method according to claim 36 which comprises afurther step of isolating the polypeptide in a substantially pure form.38. A method of preparing a baked product having improved porehomogeneity and reduced average pore diameter, the method comprisingadding the polypeptide according to claim
 1. 39. A method according toclaim 38 wherein the dough does not contain added lipids.
 40. A methodaccording to claim 38, comprising adding to the dough the polypeptide inan amount that results in a reduction of the average pore diameter inthe crumb of the bread made from the dough by at least 10%, relative toa bread which is made from a bread dough without addition of thepolypeptide.
 41. A method according to claim 38, comprising adding tothe dough the polypeptide in an amount that results in an increase ofthe pore homogeneity in the crumb of the bread made from the dough by atleast 5%, relative to a bread which is made from a bread dough withoutaddition of the polypeptide.
 42. A method according to claim 38,comprising adding to the dough the polypeptide in an amount that resultsin an increase of the gluten index in the dough of at least 5%, relativeto a dough without addition of the polypeptide, the gluten index beingdetermined by means of a Glutomatic 2200 apparatus.
 43. A methodaccording to claim 38 wherein the polypeptide is added to the dough inan amount which is in the range of 5,000-30,000 lipase units (LUS) perkg flour.
 44. A method according to claim 38 wherein an emulsifier isadded to the dough.
 45. A method of improving the stability of a glutennetwork in a dough, imparting improved pore homogeneity, reducing porediameter of a baked product made from the dough or a combinationthereof, comprising adding to the dough a polypeptide according to claim1 or a polypeptide prepared by a process according to claim
 36. 46. Amethod according to claim 45 wherein the gluten index in the dough isincreased by at least 5%, relative to a dough which is made withoutaddition of the polypeptide, according to claim 1 or the polypeptideprepared by a process according to claim 36, the gluten index beingdetermined by means of a Glutomatic 2200 apparatus.
 47. A doughimproving composition comprising the polypeptide according to claim 1and at least one further conventional dough additive component.
 48. Arecombinant DNA molecule comprising a nucleotide sequence coding for apolypeptide exhibiting lipase activity and which polypeptide comprisesat least one of the amino acid sequences shown herein as SEQ ID NO:1,SEQ ID No:2 and SEQ ID NO:3 or a nucleotide sequence coding for apolypeptide exhibiting lipase activity which comprises the amino acidsequence shown as SEQ ID No.
 9. 49. A recombinant DNA moleculecomprising at least one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQID NO:7 or at least the coding sequence of the nucleotide sequence shownas SEQ ID NO:8 or a variant, homologue or fragment thereof, or asequence complementary thereto.